Clonal amplification of nucleic acid on solid surface with template walking

ABSTRACT

Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. Non-provisionalSer. No. 14/692,706, filed Apr. 21, 2015, which is a Continuationapplication of U.S. Non-provisional Ser. No. 13/328,844, filed Dec. 16,2011 (now, abandoned), which claims the benefit of priority under 35U.S.C. § 119 to U.S. Provisional App. No. 61/552,660, filed Oct. 28,2011, and 61/526,478, filed Aug. 23, 2011, and 61/451,919, filed Mar.11, 2011, and 61/445,324, filed Feb. 22, 2011, and 61/424,599, filedDec. 17, 2010; this application is also a Continuation-in-Part of U.S.Non-provisional Ser. No. 13/828,049, filed Mar. 14, 2013 (now U.S. Pat.No. 9,309,566), which claims priority to U.S. Provisional App. No.61/692,830, filed Aug. 24, 2012, which U.S. Ser. No. 13/828,049 is aContinuation-in-Part of U.S. Ser. No. 13/328,844; this application isalso a Continuation-in-Part of U.S. Non-provisional Ser. No. 14/789,922,filed Jul. 1, 2015, which is a Continuation application of U.S.Non-provisional Ser. No. 14/085,727, filed Nov. 20, 2013 (now U.S. Pat.No. 9,309,558), which is a Continuation application of U.S.Non-provisional Ser. No. 13/923,232, filed Jun. 20, 2013 (now U.S. Pat.No. 9,371,557), which is a Continuation application of U.S.Non-provisional Ser. No. 13/842,296, filed Mar. 15, 2013 (now U.S. Pat.No. 9,309,557), which claims the benefit of priority under 35 U.S.C. §119 to U.S. Provisional App. No. 61/699,810, filed Sep. 11, 2012;61/767,766, filed Feb. 21, 2013; 61/792,247, filed Mar. 15, 2013;61/822,226, filed May 10, 2013; 61/822,239, filed May 10, 2013;61/859,000, filed Jul. 26, 2013; 61/876,136 filed Sep. 10, 2013; and62/064,283, filed Oct. 15, 2014; and this application is also aContinuation-in-Part of U.S. Non-provisional Ser. No. 14/085,718, filedNov. 20, 2013 (now abandoned), which is a Continuation application ofU.S. Non-provisional Ser. No. 13/923,232, filed Jun. 20, 2013 (now U.S.Pat. No. 9,371,557); each of which is incorporated by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 27, 2012, isnamed LT00406.txt and is 13,377 bytes in size.

REFERENCE TO BIOLOGICAL SEQUENCE DISCLOSURE

This application contains nucleotide sequence and/or amino acid sequencedisclosure in computer readable form and a written sequence listing, theentire contents of both of which are expressly incorporated by referencein their entirety as though fully set forth herein.

BACKGROUND

Nucleic acid amplification is very useful in molecular biology and haswide applicability in practically every aspect of biology, therapeutics,diagnostics, forensics and research. Generally, multiple amplicons aregenerated from a starting template using one or more primers, where theamplicons are homologous or complementary to the template from whichthey were generated. Multiplexed amplification can also streamlineprocesses and reduce overheads. A single set of primers can be mixedwith different templates, or a single template can be contacted withmultiple different primers, or multiple different templates can becontacted with multiple different primers. This application relates tomethods and reagents for nucleic acid amplification and/or analysis.

SUMMARY

Methods, reagents and products of nucleic acid amplification and/oranalysis are provided herein. Amplification can make use of immobilizedand/or soluble primers. Amplicons generated from methods provided hereinare suitable substrates for further analysis, e.g., sequencedetermination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic showing an embodiment of template walking. In analternative embodiment, the immobilized primer comprises anadenosine-rich sequence designated as (A)_(n), e.g., (A)₃₀, (SEQ IDNO:1) and the primer binding site for the immobilized primer on thetemplate comprises a complementary T-rich sequence, e.g., (T)₃₀ (SEQ IDNO:2).

FIG. 2: Overview of amplification on beads by template walking anddeposition of beads onto a planar array for sequencing

FIG. 3: Alternative embodiments using semiconductor-based detection ofsequencing by synthesis. Template walking can be used to generate apopulation of clonal amplicons on a bead or on the base or bottom of areaction chamber. In an alternative embodiment, the immobilized primercomprises an adenosine-rich sequence designated as (A)_(n), e.g., (A)₃₀(SEQ ID NO:1), and the primer binding site for the immobilized primer onthe template comprises a complementary T-rich sequence, e.g., (T)₃₀ (SEQID NO:2).

FIG. 4: Alternative embodiments of immobilization sites in the form ofprimer lawns onplanar substrates. Arrays of separated immobilizationsites can be used or else a single continuous lawn of primers can beconsidered to be a random array of immobilization sites. Optionally, thelocation of one or more immobilization sites in the continuous lawn ofprimers can be undetermined as yet, where the location is determined atthe time of attachment of the initial template before walking or isdetermined by the space occupied by the amplified cluster. In analternative embodiment, the immobilized primer comprises anadenosine-rich sequence designated as (A)_(n), e.g., (A)₃₀ (SEQ IDNO:1), and the primer binding site for the immobilized primer on thetemplate comprises a complementary T-rich sequence, e.g., (T)₃₀ (SEQ IDNO:2).

FIG. 5: Influence of temperature on the template walking reaction. Agraphical plot of the delta Ct before and after the template walkingamplification was calculated and plotted against reaction temperature.

FIG. 6: Table of the Ct values of the 96 duplex TaqMan qPCR reactions.

FIG. 7: 100,000-fold.amplification by template walking on beads. DeltaCt before and after the template walking reaction and fold ofamplification before and after the template walking reaction wascalculated and plotted against reaction time.

FIG. 8A: Schematic depiction of exemplary template walking. FIG. 8B:Schematic decpition of exemplary strand flipping to generate flippedstrands. FIG. 8C: Schmatic depiction of exemplary addition of newprimer-binding sequence Pg′ on final flipped strands. FIGS. 8A-8Cdisclose “(A)₃₀,” “T₃₀,” “(T)₂₀,” and “(T)₃₂” as SEQ ID NOS 1-3 and 22,respectively.

DEFINITIONS

Any active verb (or its gerund) is intended to indicate that thecorresponding action occurs at a specific, significant or substantiallevel (e.g., more than random, or more than an appropriate control). Forexample the act of “hybridizing” indicates that a significant orsubstantial level of specific hybridization takes place. For example inthe case of hybridization of a primer to a template during anamplification process, hybridizing is optionally sufficient to achieve adesired fold of amplification—e.g., at least 10³ or 10⁴ or 10⁵ or 10⁶amplicons from a single template. In another example, specifichybridization is more than would happen between two nucleic acids thatshare no significant amount of sequence homology. In yet anotherexample, specific hybridization comprises binding of a nucleic acid to atarget nucleotide sequence in the absence of substantial binding toother nucleotide sequences present in the hybridization mixture underdefined stringency conditions. Optionally, the sample is derived fromtissues or fluids taken from living or dead organisms (e.g., humans) forthe purposes of diagnostics or forensics). Optionally, the samecomprises a genomic library or an exome library.

Two sequences can be considered to be complementary if one sequencehybridizes substantially and specifically under the conditions of choiceto the other sequence. Two sequences can be considered to be homologousif the reverse complement of one sequence can hybridize substantiallyand specifically under the conditions of choice to the other sequence.Substantial hybridization for example is where more than 5%, optionally10%, 30%, 50% or 80% of one of the nucleic acids is hybridized to theother nucleic acid. Hybridization between two single-stranded nucleicacids often but not necessarily involves the formation of a doublestranded structure that is stable under conditions of choice. Theconditions of choice are for example conditions in which hybridizationis intended, e.g., during an annealing step of an amplification cycle.Two single-stranded polynucleotides are optionally considered to behybridized if they are bonded to each other by two or more sequentiallyadjacent base pairings. Optionally, a substantial proportion ofnucleotides in one strand of the double stranded structure undergoWatson-Crick base-pairing with a nucleoside on the other strand.Hybridization also includes the pairing of nucleoside analogs, such asdeoxyinosine, nucleosides with 2-aminopurine bases, and the like, thatmay be employed to reduce the degeneracy of the probes, whether or notsuch pairing involves formation of hydrogen bonds.

A nucleic acid can be considered immobilized if it is attached to asupport in a manner that is substantially stable, at least duringconditions of choice (e.g., during the amplification reaction). Theattachment can be by any mechanism, including but not limited tonon-covalent bonding, ionic interactions, covalent linkage. If a firstnucleic acid is hybridized to a second nucleic acid immobilized on asupport, then the first nucleic acid can also be considered to beimmobilized to the support during amplification, if amplificationconditions are such that substantial amounts of the first and secondnucleic acids are associated or connected with each other at any or alltimes during amplification. For example the first and second nucleicacids can be associated together by hybridization involving Watson-Crickbase pairing or hydrogen bonding. In an example, the amplificationconditions of choice allow at least 50%, 80%, 90%, 95% or 99% of thefirst nucleic acid to remain hybridized with the second nucleic acid, orvice versa. A nucleic acid can be considered unimmobilized ornon-immobilized if it is not directly or indirectly attached to orassociated with a support.

A medium can be considered flowable under conditions of choice if themedium is under those conditions at least temporarily a fluid mediumthat does not substantially or completely restrain or impede transfer ormovement of an unimmobilized molecule. The unimmobilized molecule is notitself immobilized to a solid support or surface or associated withanother immobilized molecule. In an embodiment, the unimmobilizedmolecule is a solute (e.g., a nucleic acid) through the flowable medium.Exemplary transfer or movement in the medium can be by means ofdiffusion, convection, turbulence, agitation, Brownian motion,advection, current flows, or other molecular movements within theliquid) from any first point in the continuous phase to any other pointin fluid communication or in the same continuous phase. For example, ina flowable medium a significant amount of an unimmobilized nucleic acidis transferred from one immobilization site to another immobilizationsite that is within the same continuous phase of the flowable medium, orin fluid communication with the first immobilization site. Optionally,the rate of transfer or movement of the nucleic acid in the medium iscomparable to the rate of transfer or movement of the nucleic acid inwater. In some instances, the conditions of choice are conditions thatthe medium is subjected to during amplification. The conditions ofchoice may or may not allow the flowable medium to remain substantiallymotionless. The conditions may or may not subject the flowable medium toactive mixing, agitation or shaking. The medium is optionally flowableat least temporarily during amplification. For example the medium isflowable under at least one preamplification and/or amplificationcondition of choice. Optionally, a flowable medium does notsubstantially prevent intermingling of different unimmobilized nucleicacids or transfer of an unimmobilized nucleic acid between differentzones of a continuous phase of the flowable medium. The movement ortransfer of nucleic acids for example can be caused by means ofdiffusion or convection. A medium is optionally considered nonflowableif unimmobilized nucleic acids upon amplification fail to spread or movebetween different immobilization sites or over the entire continuousphase. Generally, a flowable medium does not substantially confineunimmobilized nucleic acids (e.g., the templates or amplicons) withinlimited zones of the reaction volume or at fixed locations during theperiod of amplification. Optionally, a flowable medium can be renderednon-flowable by various meansor by varying its conditions. Optionally, amedium is flowable if it is liquid or is not semisolid. A medium can beconsidered flowable if its fluidity is comparable to pure water. Inother embodiments, a medium can be considered flowable if it a fluidthat is substantially free of polymers, or if its viscosity coefficientis similar to that of pure water.

In an embodiment, a support comprises one or more immobilization sites.A nucleic acid (e.g., a template or an amplicon) optionally associateswith the support at its corresponding immobilization site. Animmobilization site optionally comprises a specific portion or area of asupport, or a position or location on a support. An immobilization sitecan have or optionally can lack any specific or predetermined location,position, dimension, size, area or structure on the support, or anyother distinguishing parameter or charateristic. Optionally, one or moreof such parameters are determined for an immobilization site at the timeof association of the template with the support, and/or during or at thecompletion of amplification. In an embodiment, one ortemplate-attachment moieties (e.g., amplification primers) are attachedto the support, which optionally are or are not arranged on the supportat pre-determined or defined positions or angles or distances from thecenter of a defined point. Optionally, an arrangement of templatemolecules over one or more immobilization sites on a support is or isnot achieved through an intentional design or placement of individualattachment moieties. Such a “randomly-patterned” or “random” array ofattachment moieties (e.g., primers) can be achieved by dropping,spraying, plating or spreading a solution, emulsion, aerosol, vapor ordry preparation comprising a pool of nucleic acid molecules onto asupport and allowing the nucleic acid molecules to settle onto thesupport, with or without any intervention that would direct them tospecific or predetermined immobilization sites thereon.

A support or immobilization site optionally comprises one or moreoligonucleotides (e.g., amplification primers) attached to a support.Preferably but not necessarily, the attachment of the oligonucleotide tothe support or immobilization site is stable during subsequentamplification or other assays (e.g., more than 10%, 30%, 50%, 70% or 90%of oligonucleotides remain attached after being subjected to theamplification conditions of choice).

Optionally, in any method described herein, all primers on at least onesupport or immobilization site comprise the same sequence. The supportor immobilization site can optionally comprise other nucleic acids whichdo not hybridize to one strand of the template of interest or itscomplement. The support or immobilization site optionally does notcomprise any other nucleic acid which hybridizes to one strand of thetemplate of interest or its complement (i.e., the immobilization siteoptionally lacks any other primers). Optionally, a support orimmobilization site comprises a plurality of primers having at least twodifferent sequences. Optionally, the support or immobilization sitecomprises a species of immobilized primers that is complementary to afirst portion of a single-stranded template, and does not comprise animmobilized primer that is homologous to a second non-overlappingportion of the template (or can hybridize to the template-complement).The two portions are non-overlapping if they do not contain anysubportions that hybridize to each other or to a complement of the otherportion.

Two or more different types of primer (e.g., different in sequence) canbe present in substantially the same concentrations as one another, oralternatively in different concentrations. Optionally, the primers aresubstantially homogeneously dispersed over the support or immobilizationsite. Optionally, in any method described herein, two differentimmobilization sites are spatially separated subcomponents (e.g.,portions or areas) of a single support and/or are on different (e.g.,structurally disconnected) supports. Optionally, in any method describedherein, at least one immobilization site includes the entire surface ofthe support or the entire volume of a support. The immobilized primersare optionally uniformly distributed over the one or more supports orimmobilization sites.

Optionally, in any method described herein, the two differentimmobilization sites are located in a predetermined arrangement in or ona shared support (e.g. in a grid pattern). In other embodiments, themetes, bounds or positioning of one or more immobilization sites is notknown or predetermined before immobilization of the template to thesupport. In an example, the support comprises multiple immobilizedprimers, and the primer to which a starting template hybridizes (e.g.,before any amplification occurs) can be considered to be included within(e.g., a central point of) the immobilization site for that template. Insuch an embodiment, the positioning of an immobilization site isdetermined during or after hybridization of the primer to the template.The metes and bounds of an immobilization site can also be determinedduring or after extension and/or amplification.

A population of nucleic acids is considered clonal if a substantialportion of its members have substantially identical (or substantiallycomplementary) sequence. It will be understood that members of apopulation need not be 100% identical or complementary, e.g., a certainnumber of “errors” may occur during the course of synthesis. In anembodiment, at least 50% of the members of a population aresubstantially identical (or complementary) to each other or to areference nucleic acid molecule (i.e., a molecule of defined sequenceused as a basis for a sequence comparison). More preferably at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least99%, or more of the members of a population are substantially identical(or complementary) to the reference nucleic acid molecule. Two moleculescan be considered substantially identical if the percent identitybetween the two molecules is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%,99.9% or greater, when optimally aligned. Two molecules can beconsidered substantially complementary if the percent complementaritybetween the two molecules is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%,99.9% or greater, when optimally aligned. In addition, a low orinsubstantial level of mixing of non-homologous nucleic acids may occurduring methods described herein, and thus a clonal population maycontain a minority of diverse nucleic acids (e.g., less than 30%, e.g.,less than 10%).

As will be appreciated by one of ordinary skill in the art, referencesto templates, initializing oligonucleotides, extension probes, primers,etc., can refer to populations or pools of nucleic acid molecules thatare substantially identical within a relevant portion, rather thansingle molecules. For example, a “template” can refer to a plurality ofsubstantially identical template molecules; a “probe” can refer to aplurality of substantially identical probe molecules, etc. In the caseof probes that are degenerate at one or more positions, it will beappreciated that the sequence of the probe molecules that comprise aparticular probe will differ at the degenerate positions, i.e., thesequences of the probe molecules that constitute a particular probe maybe substantially identical only at the nondegenerate position(s). Theseterms within this application are intended to provide support for eithera population or a molecule. Where it is intended to refer to a singlenucleic acid molecule (i.e., one molecule), the terms “templatemolecule”, “probe molecule”, “primer molecule”, etc., may be usedinstead. In certain instances the plural nature of a population ofsubstantially identical nucleic acid molecules will be explicitlyindicated.

“Template”, “oligonucleotide”, “probe”, “primer”, “template”, “nucleicacid” and the like are intended to be interchangeable terms herein.These terms refer to polynucleotides, not necessarily limited to anylength or function. The same nucleic acid can be regarded as a“template”, “probe” or “primer” depending on the context, and can switchbetween these roles with time. A “polynucleotide,” also called a“nucleic acid,” is a linear polymer of two or more nucleotides joined bycovalent internucleosidic linkages, or variant or functional fragmentsthereof. In naturally occuring examples of these, the internucleosidelinkage is typically a phosphodiester bond. However, other examplesoptionally comprise other internucleoside linkages, such asphosphorothiolate linkages and may or may not comprise a phosphategroup. Polynucleotides include double- and single-stranded DNA, as wellas double- and single-stranded RNA, DNA:RNA hybrids, peptide-nucleicacids (PNAs) and hybrids between PNAs and DNA or RNA, and also includeknown types of modifications. Polynucleotides can optionally be attachedto one or more non-nucleotide moieties such as labels and other smallmolecules, large molecules such proteins, lipids, sugars, and solid orsemi-solid supports, for example through either the 5′ or 3′ end. Labelsinclude any moiety that is detectable using a detection method ofchoice, and thus renders the attached nucleotide or polynucleotidesimilarly detectable using a detection method of choice. Optionally, thelabel emits electromagnetic radiation that is optically detectable orvisible. In some cases, the nucleotide or polynucleotide is not attachedto a label, and the presence of the nucleotide or polynucleotide isdirectly detected. A “nucleotide” refers to a nucleotide, nucleoside oranalog thereof. Optionally, the nucleotide is an N- or C-glycoside of apurine or pyrimidine base. (e.g., deoxyribonucleoside containing2-deoxy-D-ribose or ribonucleoside containing D-ribose). Examples ofother analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides. Referring to a nucleic acid by any one ofthese terms should not be taken as implying that the nucleic acid hasany particular activity, function or properties. For example, the word“template” does not indicate that the “template” is being copied by apolymerase or that the template is not capable of acting as a “primer”or a “probe”.

It will be appreciated that in certain instances nucleic acid reagentsinvolved in amplification such as a template, probe, primer, etc., maybe a portion of a larger nucleic acid molecule that also containsanother portion that does not serve the same function. Optionally, thisother portion does not serve any template, probe, or primer function. Insome instances, a nucleic acid that substantially hybridizes to anoptionally-immobilized primer (e.g., on an immobilization site) isconsidered to be the “template”. Any one or more nucleic acid reagentsthat are involved in template walking (template, immobilized strands,immobilized or unimmobilized primer, etc.) may be generated before orduring amplification from other nucleic acids. The nucleic acid reagentis optionally generated from (and need not be identical to) an inputnucleic acid by making one or more modifications to the nucleic acidthat was initially introduced into the template walking medium. An inputnucleic acid can for example be subjected to restriction digestion,ligation, one or more amplification cycles, denaturation, mutation, etc,to generate a nucleic acid that serves as the template, primer, etc,during amplification or further amplification. For example, adouble-stranded input nucleic acid can be denatured to generate a firstsingle-stranded nucleic acid which optionally is used to generate asecond complementary strand. If so desired, the first single-strandednucleic acid can be considered the “template” for our purposes herein.Alternatively, the second complementary strand generated from the firstsingle-stranded nucleic acid can be considered the “template” for ourpurposes herein. In another example, a template is derived from an inputnucleic acid and is not necessarily identical to the input nucleic acid.For example, the template can comprise additional sequence not presentan input nucleic acid. In an embodiment the template can be an amplicongenerated from an input nucleic acid using one or more primers with a 5′overhang that is not complementary to the input nucleic acid.

The term “amplifying” refers to production of copies of a nucleic acidmolecule, for example via repeated rounds of primed enzymatic synthesis.Optionally, such amplifying takes place with an immobilized templatenucleic acid molecule and/or one or more primers that are immobilized.An amplicon is for example a single-stranded or double-stranded nucleicacid that is generated by an amplification procedure from a startingtemplate nucleic acid. The amplicon comprises a nucleic acid strand, ofwhich at least a portion is substantially identical or substantiallycomplementary to at least a portion of the starting template. Where thestarting template is double-stranded, an amplicon comprises a nucleicacid strand that is substantially identical to at least a portion of onestrand and is substantially complementary to at least a portion ofeither strand. The amplicon can be single-stranded or double-strandedirrespective of whether the initial template is single-stranded ordouble-stranded.

The term “support” includes any solid or semisolid article on whichreagents such as nucleic acids can be immobilized. Nucleic acids may beimmobilized on the solid support by any method including but not limitedto physical adsorption, by ionic or covalent bond formation, orcombinations thereof. A solid support may include a polymeric, a glass,or a metallic material. Examples of solid supports include a membrane, aplanar surface, a microtiter plate, a bead, a filter, a test strip, aslide, a cover slip, and a test tube. means any solid phase materialupon which a oligomer is synthesized, attached, ligated or otherwiseimmobilized. A support can optionally comprise a “resin”, “phase”,“surface” and “support”. A support may be composed of organic polymerssuch as polystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A support may also be inorganic, such as glass, silica,controlled-pore-glass (CPG), or reverse-phase silica. The configurationof a support may be in the form of beads, spheres, particles, granules,a gel, or a surface. Surfaces may be planar, substantially planar, ornon-planar. Supports may be porous or non-porous, and may have swellingor non-swelling characteristics. A support can be shaped to comprise oneor more wells, depressions or other containers, vessels, features orlocations. A plurality of supports may be configured in an array atvarious locations. A support is optionally addressable (e.g., forrobotic delivery of reagents), or by detection means including scanningby laser illumination and confocal or deflective light gathering. Anamplification support (e.g., a bead) can be placed within or on anothersupport (e.g., within a well of a second support).

In an embodiment the solid support is a “microparticle,” “bead”“microbead”, etc., (optionally but not necessarily spherical in shape)having a smallest cross-sectional length (e.g., diameter) of 50 micronsor less, preferably 10 microns or less, 3 microns or less, approximately1 micron or less, approximately 0.5 microns or less, e.g., approximately0.1, 0.2, 0.3, or 0.4 microns, or smaller (e.g., under 1 nanometer,about 1-10 nanometer, about 10-100 nanometers, or about 100-500nanometers). Microparticles (e.g., Dynabeads from from Dynal, Oslo,Norway) may be made of a variety of inorganic or organic materialsincluding, but not limited to, glass (e.g., controlled pore glass),silica, zirconia, cross-linked polystyrene, polyacrylate,polymehtymethacrylate, titanium dioxide, latex, polystyrene, etc.Magnetization can facilitate collection and concentration of themicroparticle-attached reagents (e.g., polynucleotides or ligases) afteramplification, and can also facilitate additional steps (e.g., washes,reagent removal, etc.). In certain embodiments of the invention apopulation of microparticles having different shapes sizes and/or colorscan be used. The microparticles can optionally be encoded, e.g., withquantum dots such that each microparticle can be individually oruniquely identified.

DETAILED DESCRIPTION

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for clonally amplifying oneor more nucleic acid templates to form clonally amplified populations ofnucleic acid templates. Any amplification method described hereinoptionally comprises repeated cycles of nucleic acid amplification. Acycle of amplification optionally comprises (a) hybridization of primerto a template strand, (b) primer extension to form a first extendedstrand, (c) partial or incomplete denaturation of the extended strandfrom the template strand. The primer that hybridizes to the templatestrand (designated “forward” primer for convenience) is optionallyimmobilized on or to a support. The support is for example solid orsemi-solid. Optionally, the denatured portion of the template strandfrom step (c) is free to hybridize with a different forward primer inthe next amplification cycle. In an embodiment, primer extension in asubsequent amplification cycle involves displacement of the firstextended strand from the template strand. A second “reverse” primer canfor example be included which hybridizes to the 3′ end of the firstextended strand. The reverse primer is optionally not immobilized.

In an embodiment, the templates are amplified using primers immobilizedon/to one or more solid or semi-solid supports. Optionally the supportcomprises immobilized primers that are complementary to a first portionof a template strand. Optionally, the support does not significantlycomprise an immobilized primer that is homologous to a secondnon-overlapping portion of the same template strand. The two portionsare non-overlapping if they do not contain any subportions thathybridize to each other or to a complement thereof. In another example,the support optionally does not significantly comprise an immobilizedprimer that can hybridize to the complement of the template strand).

Optionally, a plurality of nucleic acid templates are amplifiedsimultaneously in a single continuous liquid phase in the presence ofone or more supports, where each support comprises one or moreimmobilization sites. In an embodiment, each template is amplified togenerate a clonal population of amplicons, where individual clonalpopulations are immobilized within or on a different support orimmobilization site from other amplified populations. Optionally, theamplified populations remain substantially clonal after amplification.

A template is for example amplified to generate clonal populations whichcomprise template-homologous strands (called “template strands” or“reverse strands” herein) and/or template-complementary strands (called“primer strands” or “forward strands” herein). In an embodimentclonality is maintained in the resulting amplified nucleic acidpopulations by maintaining association between template strands and itsprimer strands, thereby effectively associating or “tethering”associated clonal progeny together and reducing the probability ofcross-contamination between different clonal populations. Optionally,one or more amplified nucleic acids in the clonal population is attachedto a support. A clonal population of substantially identical nucleicacids can optionally have a spatially localized or discrete macroscopicappearance. In an embodiment a clonal population can resemble a distinctspot or colony (e.g., when distributed in a support, optionally on theouter surface of the support).

In some embodiments, the disclosure relates generally to novel methodsof generating a localized clonal population of clonal amplicons,optionally immobilized in/to/on one or more supports. The support canfor example be solid or semisolid (such as a gel or hydrogel). Theamplified clonal population is optionally attached to the support'sexternal surface or can also be within the internal surfaces of asupport (e.g., where the support has a porous or matrix structure).

In some embodiments, amplification is achieved by multiple cycles ofprimer extension along a template strand of interest (also called a“reverse” strand). For convenience, a primer that hybridizes to thetemplate strand of interest is termed a “forward” primer, and isoptionally extended in template-dependent fashion to form a “forward”strand that is complementary to the template strand of interest. In somemethods, the forward strand is itself hybridized by a second primertermed the “reverse” primer, which is extended to form a new templatestrand (also called a reverse strand). Optionally, at least a portion ofthe new template strand is homologous to the original template(“reverse”) strand of interest.

As mentioned, one or more primers can be immobilized in/on/to one ormore supports. Optionally, one primer is immobilized by attachment to asupport. A second primer can be present and is optionally notimmobilized or attached to a support. Different templates can forexample be amplified onto different supports or immobilization sitessimultaneously in a single continuous liquid phase to form clonalnucleic acid populations. A liquid phase can be considered continuous ifany portion of the liquid phase is in fluid contact or communicationwith any other portion of the liquid body. In another example, a liquidphase can be considered continuous if no portion is entirely subdividedor compartmentalized or otherwise entirely physically separated from therest of the liquid body. Optionally, the liquid phase is flowable.Optionally, the continuous liquid phase is not within a gel or matrix.In other embodiments, the the continuous liquid phase is within a gel ormatrix. For example the continuous liquid phase occupies pores, spacesor other interstices of a solid or semisolid support.

Where the liquid phase is within a gel or matrix, one or more primersare optionally immobilized on a support. Optionally the support is thegel or matrix itself. Alternatively the support is not the gel or matrixitself. In an example one primer is immobilized on a solid supportcontained within a gel and is not immobilized to gel molecules. Thesupport is for example in the form of a planar surface or one or moremicroparticles. Optionally the planar surface or plurality ofmicroparticles comprises forward primers having substantially identicalsequence. In an embodiment, the support does not contain significantamounts of a second different primer. Optionally, a secondnon-immobilized primer is in solution within the gel. The secondnon-immobilized primer for example binds to a template strand (i.e.,reverse strand), whereas the immobilized primer binds to a forwardstrand.

For convenience, the portion of a nucleic acid template strand that ishybridized by a primer will be referred to as the “primer-bindingsequence” or PBS. Thus, a forward primer binds to a forward-primerbinding sequence (“forward PBS”) on a reverse strand, while a reverseprimer binds to a reverse PBS on the forward strand.

An embodiment includes a method of primer extension, comprising: (a) aprimer-hybridization step, (b) an extension step, and (c) a walkingstep. Optionally, the primer-hybridization step comprises hybridizing afirst primer molecule (“first forward primer”) to a complementaryforward-primer-binding sequence (“forward PBS”) on a nucleic acid strand(“reverse strand”). Optionally the extension step comprises generatingan extended first forward strand that is a full-length complement of thereverse strand and is hybridized thereto. The extended first forwardstrand is for example generated by extending the first forward primermolecule in template-dependent fashion using the reverse strand astemplate. Optionally the walking step comprises hybridizing a secondprimer (“second forward primer”) to the forward PBS where the reversestrand is also hybridized to the first forward strand. For example, thewalking step comprises denaturing at least a portion of the forward PBSfrom the forward strand (“free portion”), where another portion of thereverse strand remains hybridized to the forward strand.

In an embodiment, the primer extension method is an amplificationmethod, in which any one or more steps of primer-hybridization,extension and/or walking are repeated at least once. For example, themethod can comprise amplifying the forward strand by one or moreamplification cycles. An amplification cycle optionally comprisesextension and walking. An exemplary amplification cycle comprises orconsists essentially of extension followed by walking. Optionally, thesecond forward primer of a first amplification cycle acts as the firstforward primer of a subsequent amplification cycle. For example, thesecond forward primer of a walking step in a first amplification cycleacts as the first forward primer of an extension step of a subsequentamplification cycle.

Optionally, the method of primer extension or amplification furthercomprises extending or amplifying the reverse strand by (a) hybridizinga first reverse primer molecule to a complementaryreverse-primer-binding sequence (“reverse PBS”) on an extended forwardstrand; (b) generating an extended first reverse strand that is afull-length complement of the forward strand and hybridized thereto, byextending the first reverse primer molecule in template-dependentfashion using the forward strand as template; and (c) hybridizing asecond primer (“second reverse primer”) to the reverse PBS where theforward strand is also hybridized to the first reverse strand. One ormore repetitions of steps (b)-(c) are optionally performed, wherein thesecond reverse primer of step (c) is the first reverse primer ofrepeated step (b); and wherein a substantial proportion of forwardstrands are hybridized to reverse strands at all times during or betweensaid one or more repetitions. In embodiments, the substantial proportionis optionally at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or 99%.

Optionally, during amplification the reverse strand and/or forwardstrand is not exposed to totally-denaturing conditions that would resultin complete separation of a significant fraction (e.g., more than 10%,20%, 30%, 40% or 50%) of a large plurality of strands from theirextended and/or full-length complements.

In an embodiment a substantial proportion of forward and/or reversestrands are optionally hybridized to extended and/or full-lengthcomplements at all times during or between one or more amplificationcycles (e.g., 1, 5, 10, 20, or all amplification cycles performed). Inembodiments, the substantial proportion of strands is optionally atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of strands. In anembodiment this is achieved by maintaining the amplification reaction ata temperature higher than the T_(m) of unextended primers, but lowerthan the T_(m) of the primer-complementary strands. For example,amplification conditions are kept within a temperature that is higherthan the T_(m) of unextended forward primers, but lower than the T_(m)of extended or full-length reverse strands. Also for example,amplification conditions are kept within a temperature that is higherthan the T_(m) of unextended reverse primers, but lower than the T_(m)of extended or full-length forward strands.

Optionally, one or more forward primers, and/or one or more reverseprimers are breathable, e.g., have a low T_(m). In an example at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of nucleotide bases of abreathable primer are adenine, thymine or uracil or are complementary toadenine, thymine or uracil.

The T_(m) of a nucleic acid strand (e.g., a primer or template strand)is for example the temperature at which at least a desired fraction of aclonal population of duplexes are rendered completely single-strandedunder the chosen reagent conditions, where an individual duplexcomprises the nucleic acid strand in question hybridized to itsfull-length complement. By default, the desired fraction is 50%. Inembodiments, the desired fraction is optionally at least 10%, 20%, 30%,40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. In anembodiment the T_(m)(N) is theoretically predicted using known methods,e.g., as discussed herein. In another embodiment the T_(m) isempirically measured by known methods. (e.g., Spink, Methods Cell Biol.2008; 84:115-41; Meunier-Prest et al., Nucleic Acids Res. 2003 Dec. 1;31(23): e150; Brewood et al., Nucleic Acids Res. 2008 September; 36(15):e98.)

The T_(m) for a desired fraction can be depicted as T_(m)(N) where Ndenotes the desired fraction in percentage terms. In an embodiment theT_(m)(50) of the forward and/or reverse primer (at which 50% of allprimer molecules are completely dissociated from their complementaryPBSs) is not more than 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or80° C. Optionally, the T_(m)(50) of the extended or full-length forwardand/or reverse strands (at which 50% of all strand molecules arecompletely dissociated from their complementary sequences) is not lessthan 80° C., 75° C., 70° C., 65° C., 60° C. or 55° C. In anotherembodiment the T_(m)(80) of the forward and/or reverse primer is notmore than 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C.Optionally, the T_(m)(80) of the extended or full-length forward and/orreverse strands is not less than 80° C., 75° C., 70° C., 65° C., 60° C.or 55° C. In another embodiment the T_(m)(90) of the forward and/orreverse primer is not more than 50° C., 55° C., 60° C., 65° C., 70° C.,75° C. or 80° C. Optionally, the T_(m)(90) of the extended orfull-length forward and/or reverse strands is not less than 80° C., 75°C., 70° C., 65° C., 60° C. or 55° C. In another embodiment the T_(m)(95)of the forward and/or reverse primer is not more than 50° C., 55° C.,60° C., 65° C., 70° C., 75° C. or 80° C. Optionally, the T_(m)(95) ofthe extended or full-length forward and/or reverse strands is not lessthan 80° C., 75° C., 70° C., 65° C., 60° C. or 55° C. Optionally, theT_(m)(99) of the extended or full-length forward and/or reverse strandsis not less than 80° C., 75° C., 70° C., 65° C., 60° C. or 55° C. Inanother embodiment the T_(m)(95) of the forward and/or reverse primer isnot more than 50° C., 55° C., 60° C., 65° C., 70° C., 75° C. or 80° C.Optionally, the T_(m)(99) of the extended or full-length forward and/orreverse strands is not less than 80° C., 75° C., 70° C., 65° C., 60° C.or 55° C.

Optionally, one or more amplification cycles (e.g., 1, 5, 10, 20, orsubstantially all amplification cycles) are performed at temperaturethat is higher than the T_(m)(70) of an unextended primer and lower thanthe T_(m)(20) of the complementary full-length strand. For example, thetemperature is higher than the T_(m)(80) of an unextended primer andlower than the T_(m)(20) or T_(m)(15) or T_(m)(10) or T_(m)(5) orT_(m)(1) of the complementary full-length strand. Also for example thetemperature is higher than the T_(m)(90) of an unextended primer andlower than the T_(m)(20) or T_(m)(15) or T_(m)(10) or T_(m)(5) orT_(m)(1) of the complementary full-length strand. Also for example thetemperature is higher than the T_(m)(95) of an unextended primer andlower than the T_(m)(20) or T_(m)(15) or T_(m)(10) or T_(m)(5) orT_(m)(1) of the complementary full-length strand. Also for example thetemperature is higher than the T_(m)(98) of an unextended primer andlower than the T_(m)(20) or T_(m)(15) or T_(m)(10) or T_(m)(5) orT_(m)(1) of the complementary full-length strand. Also for example thetemperature is higher than the T_(m)(99) of an unextended primer andlower than the T_(m)(20) or T_(m)(15) or T_(m)(10) or T_(m)(5) orT_(m)(1) of the complementary full-length strand. Optionally, the one ormore amplification cycles are performed at temperature that is at least5, 10, 15, 20, 25, 30, 35, or 45° C. higher than the T_(m)(50) of anunextended primer. Optionally, the temperature is at least 5, 10, 15,20, 25, 30, 35, or 45° C. lower than the T_(m)(50) of a full-lengthprimer-complementary strand. In an embodiment the unextended primer is aforward primer and the complementary full-length strand is a reversestrand, or vice versa.

Optionally, template-dependent extension of a forward primer using areverse strand as template in the extension step results in displacementof another forward strand that was already hybridized to the reversestrand. Optionally, template-dependent extension of a reverse primerusing a forward strand as template in the extension step results indisplacement of another reverse strand that was already hybridized tothe forward strand.

In an embodiment the method further comprises completely separating theextended forward strands from reverse strands after performing primerextension or a desired number of amplification cycles, and optionallyremoving separated forward strands from the presence of separatedimmobilized reverse strands, or vice versa.

Optionally, one or more nucleic acid reagents are not in contact with arecombinase and/or reverse transcriptase and/or helicase and/or nickingenzyme and/or any other enzyme that is not a polymerase during any oneor more steps. For example the primers and/or template are not incontact with one or more such enzymes at any time.

Denaturation is optionally achieved non-enzymatically, e.g., by raisingthe temperature. In an embodiment amplification is performed undersubstantially isothermal conditions, as described herein.

Optionally, any one or more nucleic acids, e.g., primers are attached(e.g., covalently attached) to a support. In an embodiment the firstand/or second forward primers are immobilized to a single (same)support.

In an embodiment, first and second forward primers are closelyimmobilized to the same support, whereby amplification generates animmobilized clonal population of extended forward strands. Optionally,the distance between the first and second forward primers is not morethan twice the length of the primer or the PBS.

Optionally, a plurality of template nucleic acids are individuallyhybridized to spatially-separated immobilization sites, wherebyamplification generates spatially-separated clonal populationscorresponding to individual template nucleic acids.

In an embodiment, first forward primer is hybridized to a forward PBS ona “reverse” template strand. Optionally, the first forward primer isimmobilized on a support. The first forward primer can be extended alongthe reverse strand to form (an extended) first forward strand. Theextension is optionally template-dependent, using the reverse strand asa template. After extension, the first forward strand and the reversestrand are optionally hybridized to each other in a duplex. Optionally,at least part of the PBS of the first reverse strand and theforward-primer portion of the first forward strand are separated fromone another (e.g., via denaturation or melting), but the first reversestrand and the first forward strand remain associated with (e.g.,hybridized to) each other over another portion. The separated portion ofthe reverse strand including at least part of the forward-PBS can thenbe annealed (e.g., by hybridization) to a second, different forwardprimer. Optionally, the second forward primer is immobilized on asupport. The second forward primer can for example be immobilized on thesame support as the first forward primer, and is optionally situatedsufficiently close to the first primer so that the a portion of thereverse strand can hybridize to the first forward strand while anotherportion of the reverse strand is hybridized to the second forward primersimultaneously. The second forward primer can then be extended in turnto form an extended second forward strand. Optionally, the secondforward primer is extended along the reverse strand in atemplate-dependent fashion. Extension of the second forward primeroptionally displaces the first forward strand from the reverse strand.As before, the primer binding sequence of the reverse strand can beseparated from the primer portion of the second forward strand, whereanother portion of the reverse strand remains associated (e.g., byhybridization) with the second forward strand. These steps can berepeated to with further forward primers to form further extendedforward strands (for example, extended third, fourth, fifth, sixth,seventh, eighth, ninth, tenth or higher order extended strands). Theprocess can optionally be repeated for a desired number of amplificationcycles to provide a population of amplified, immobilized nucleic acidmolecules. The population can be substantially clonal in nature. Forexample, the amplified nucleic acid molecules of the amplified clonalpopulation can include a plurality of nucleic acids that aresubstantially identical and/or substantially complementary to eachother.

Optionally, the extended forward strands comprise areverse-primer-binding sequence (“reverse PBS”), to which a reverseprimer hybridizes. The reverse PBS on the forward strand optionallycomprises or is near the 3′ end of the forward strands. In someembodiments, amplification involves dissociating least part of thereverse PBS from any hybridized or associated sequence, such as sequenceon a reverse strand. The reverse PBS on the forward strand is optionallycontacted with a reverse primer which hybridizes to it. The reverseprimer is then extended using the forward strand as template to form anextended reverse strand. Optionally, the newly-generated reverse strandacts as template for forward primer extension. The reverse strand canalso participate as template, primer, or probe in another reaction,including any method described herein.

The amplified nucleic acid populations can be used for many differentpurposes, including sequencing, screening, diagnosis, in situ nucleicacid synthesis, monitoring gene expression, nucleic acid fingerprinting,etc.

Optionally, any one or more primer extension and/or amplificationmethods herein generate one or more immobilized nucleic-acid extensionproducts. In a variation, a solid support comprises primers that allidentical or substantially identical. The solid support can compriseother nucleic acids. Optionally, these other nucleic acids do nothybridize to a template strand of interest or its complement. The solidsupport optionally does not comprise any other nucleic acid whichhybridizes to the template strand of interest or its complement.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for clonally amplifying anucleic acid template onto a support in an amplification reactionsolution. Optionally, the nucleic acid template is contacted with asupport in a solution comprising a continuous liquid phase. The supportcan include a population of primers, including at least a first primerand a second primer. The population of primers can be immobilized on thesupport, for example by covalent attachment to the support. In someembodiments, the nucleic acid template includes a primer bindingsequence adjacent to a target sequence. The primer binding sequence canby complementary to a sequence of the first primer and optionally asequence of the second primer. The target sequence can benoncomplementary to the primers in the population. In some embodiments,the primer binding sequence of the nucleic acid template is hybridizedto the first primer. The first primer can be extended along the templateusing a polymerase, thereby forming an extended first primer. At least aportion of the primer binding sequence of the template can be separated(e.g., denatured or melted) from the extended first primer. Theseparating is optionally performed while maintaining hybridizationbetween a portion of the template and the extended first primer. Theseparated portion of the primer binding sequence can be subsequentlyhybridized to the second primer. Optionally, such hybridization isperformed while maintaining hybridization between the other portion ofthe template and the extended first primer. The second primer can beextended along the template using a polymerase, thereby forming asupport including an extended first primer and an extended secondprimer. The extended portion of the extended first primer and/or theextended second primer can include sequence complementary to the targetsequence.

In some embodiments, the disclosure relates generally to methods forclonally amplifying a nucleic acid template onto a support in anamplification reaction solution, comprising: contacting a nucleic acidtemplate with a support in a liquid solution, wherein the supportincludes a population of immobilized primers including at least a firstprimer and a second primer, and wherein the nucleic acid templateincludes a primer binding sequence adjacent to a target sequence, wherethe primer binding sequence is complementary to a sequence of the firstprimer and a sequence of the second primer, and the target sequence isnoncomplementary to the primers in the population; hybridizing theprimer binding sequence of the nucleic acid template to the firstprimer; extending the first primer along the template using apolymerase, thereby forming an extended first primer; denaturing atleast a portion of the primer binding sequence of the template from theextended first primer while maintaining hybridization between anotherportion of the template and the extended first primer; hybridizing thedenatured portion of the primer binding sequence to the second primerwhile maintaining hybridization between the other portion of thetemplate and the extended first primer; and extending the second primeralong the template using a polymerase, thereby forming a supportincluding an extended first primer and an extended second primer, wherethe extended first primer and the extended second primer each includesequence complementary to the target sequence. The population of primerscan be comprised of substantially identical primers that differ insequence by no more than one, two, three, four or five nucleotides. Insome embodiments, the primer population is comprised of differentprimers, at least some of which include a sequence that is complementaryto the primer binding sequence of the template. In some embodiments, theprimers of population are noncomplementary to the sequence of the 5′terminal half of the template. In some embodiments, the primers of thepopulation are noncomplementary to the sequence of the 3′ terminal halfof any of the extended primers of the support. In some embodiments, theprimers of the population are noncomplementary to any sequence of thetemplate other than the primer binding sequence.

In some embodiments, the disclosure relates generally to methods forclonally amplifying a population of nucleic acid templates onto apopulation of supports in an amplification reaction solution,comprising: clonally amplifying a first template onto a first nucleicacid template onto a first support according to any of the methodsdisclosed herein, and clonally amplifying a second nucleic acid templateonto a second support according to the same method, wherein bothsupports are included within a single continuous liquid phase during theamplifying.

As will be appreciated by one of ordinary skill in the art, referencesto templates, initializing oligonucleotides, extension probes, primers,etc., can in some embodiments refer to populations or pools of nucleicacid molecules that are substantially identical within a relevant regionrather than single molecules. Thus, for example, a “template” can insome embodiments refer to a plurality of substantially identicaltemplates; a “probe” can in some embodiments refer to a plurality ofsubstantially identical probe molecules, etc. In the case of probes thatare degenerate at one or more positions, it will be appreciated that thesequence of the probe molecules that comprise a particular probe willdiffer at the degenerate positions, i.e., the sequences of the probemolecules that constitute a particular probe may be substantiallyidentical only at the nondegenerate position(s). For purposes ofdescription the singular form can be used to refer not only to singlemolecules but also to populations of substantially identical molecules.In certain instances the singular nature of a nucleic acid molecule, orthe plural nature of a population of substantially identical nucleicacid molecules, will be explicitly indicated.

It will be understood that members of a population need not be 100%identical. For example, all members of a clonally amplified populationof nucleic acid sequence need not be identical since a certain number of“errors” may occur during the course of synthesis; similarly, not allprimers within a population of primers may be identical to each other.In some embodiments, at least 50% of the members of a population areidentical to a reference nucleic acid molecule (i.e., a molecule ofdefined sequence used as a basis for a sequence comparison). In someembodiments, at least 50% of the members of a population are at least70%, 75%, 85%, 90%, or more preferably at least 95% identical to areference nucleic acid molecule. More preferably at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, or more ofthe members of a population are at least 85%, 90%, or more preferably atleast 95% identical, or yet more preferably at least 99% identical tothe reference nucleic acid molecule. Preferably the percent identity ofat least 95% or more preferably at least 99% of the members of thepopulation to a reference nucleic acid molecule is at least 98%, 99%,99.9% or greater. Percent identity may be computed by comparing twooptimally aligned sequences, determining the number of positions atwhich the identical nucleic acid base (e.g., A, T, C, G, U, or I) occursin both sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions, andmultiplying the result by 100 to yield the percentage of sequenceidentity. It will be appreciated that in certain instances a nucleicacid molecule such as a template, probe, primer, etc., may be a portionof a larger nucleic acid molecule that also contains a portion that doesnot serve a template, probe, or primer function. In that case individualmembers of a population need not be substantially identical with respectto that portion.

A nucleic acid optionally comprises one or more nucleotides. In anembodiment a nucleotide comprises any one or more of a nucleobase(nitrogenous base), a five-carbon sugar (either ribose or2′-deoxyribose), and a phosphate group. Optionally, a nucleotidecomprises all three components or derivatives thereof. Optionally, thenucleobases is a purine or a pyrimidine base. Exemplary purine basesinclude adenine and guanine, while exemplary pyrimidines are thymine,uracil and cytosine

As used herein, the term “complementary” and its variants, when used inreference to individual nucleotides, include nucleotides that areefficiently incorporated by DNA polymerases opposite each other duringDNA replication under physiological conditions. In an typicalembodiment, complementary nucleotides can form base pairs with eachother, such as the A-T/U and G-C base pairs formed through specificWatson-Crick type hydrogen bonding between the nucleobases ofnucleotides and/or polynucleotides positions antiparallel to each other;other types of base pairing can also occur. For example, thecomplementarity of other artificial base pairs can be based on othertypes of hydrogen bonding and/or hydrophobicity of bases and/or shapecomplementarity between bases.

As used herein, the term “complementary” and its variants, when used inreference to nucleic acid sequences, refers to nucleic acid sequencesthat can undergo cumulative base pairing with each other at two or moreindividual corresponding positions in antiparallel orientation, as in ahybridized duplex. Optionally there can be “complete” or “total”complementarity between a first and second nucleic acid sequence whereeach nucleotide in the first nucleic acid molecule or sequence canundergo a stabilizing base pairing interaction with a nucleotide in thecorresponding antiparallel position on the second nucleic acid sequence;alternatively, two nucleic acid sequences can be complementary when atleast 50% of the nucleotide residues of one nucleic acid sequence arecomplementary to nucleotide residues in the other nucleic acid sequence.The complementary residues within a particular complementary nucleicacid sequence need not always be contiguous with each other, and can beinterrupted by one or more noncomplementary residues within thecomplementary nucleic acid sequence. In some embodiments, at least 50%,but less than 100%, of the residues of one of the two complementarynucleic acid sequences are complementary to residues in the othernucleic acid sequence. In some embodiments, at least 70%, 80%, 90%, 95%or 99% of the residues of one nucleic acid sequence are complementary toresidues in the other nucleic acid sequence. Sequences are said to be“substantially complementary” when at least 85% of the residues of onenucleic acid sequence are complementary to residues in the other nucleicacid sequence.

As used herein, “complementary” when used in reference to two or morenucleic acid molecules, can include any nucleic acid molecules whereeach molecule comprises a sequence that is complementary to a sequencein the other nucleic acid molecules. The complementary nucleic acidmolecules need not be complementary across their entire length, and eachmolecule can include one or more regions that is noncomplementary to theother molecules. For example, a template and a primer molecule can bereferred to as “complementary” even when they are of different lengths;in some embodiments, the template can be longer than the primer andinclude sequence that is noncomplementary to any sequence of the primer,or vice versa.

The term “noncomplementary” and its variants, as used herein withreference to two nucleic acid molecules or sequences, typically refersto nucleic acid molecules or sequences in which less than 50% of theresidues of one nucleic acid molecule or sequence are complementary toresidues in the other nucleic acid molecule or sequence. A “mismatch” ispresent at any position in the nucleic acid molecule molecules orsequences where the two opposed nucleotides are not complementary.Similarly, two nucleotide sequences or portions thereof are consideredto match each other if the sequences or portions are identical orcomplementary to each other.

As used herein, the term “hybridization” refers to the process of basepairing between any two nucleic acid molecules including complementarynucleotides at one or more positions. Typically, such base pairing canoccur according to established paradigms, for example the Watson-Crickparadigm wherein A-T/U and G-C base pairs are formed through specificWatson-Crick type hydrogen bonding between the nucleobases ofnucleotides and/or polynucleotides positions antiparallel to each other.In some embodiments, hybridization can occur according to non-WatsonCrick paradigms as well; for example, artificial base pairs can beformed through other types of hydrogen bonding and/or hydrophobicity ofbases and/or shape complementarity between bases. The hybridized nucleicacid molecules need not be hybridized across their entire length, andeach molecule can include one or more regions not hybridized to theother molecule. For example, a template and a primer molecule can bedescribed as hybridized to each other even when a substantial region ofthe template, primer or both remain non-hybridized to each other.Furthermore, a region of hybridization can include one or morecontiguous nucleotides that are not base paired with each other. Nucleicacid molecules (or sequences within nucleic acid molecules) that arebase paired with each other in this manner are referred to as“hybridized”. In some embodiments, a single nucleic acid molecule mayundergo self-hybridization (e.g., hairpin formation) with itself.

Typically, hybridizing nucleic acid molecules (for example, hybridizinga primer with a template) includes contacting nucleic acid moleculeswith each other under conditions where one or more nucleotide residueswithin each nucleic acid molecule base pairs with one or morenucleotides of another nucleic acid molecule. The contacting can beperformed using any suitable conditions, depending on the desiredapplication. In one exemplary assay, two nucleic acid molecules arecontacted in a buffered solution comprising salts and/or detergents,e.g., SDS, for a desired length of time and for a desired period. Forexample, the hybridization can be performed using low stringency, mediumstringency or high stringency hybridization conditions. The stringencyof hybridization can be adjusted by varying various hybridizationparameters, including for example temperature, salt concentration, SDSconcentration, at the like. Methods of hybridization and of controllingthe stringency of hybridization are well known in the field.

Among other things, a method is provided of generating a localizedclonal population of immobilized clonal amplicons of a single-strandedtemplate sequence, comprising: (a) attaching the single-strandedtemplate sequence (“template 1”) to an immobilization site (“IS1”),wherein IS1 comprises multiple copies of an immobilized primer (“IS1primer”) which can hybridize substantially to template 1, and template 1is attached to IS1 by hybridization to an IS1 primer, and (b) amplifyingtemplate 1 using IS1 primer and a non-immobilized primer (“SP1 primer”)in solution, wherein amplified strands that are complementary to thesingle-stranded template 1 cannot hybridize substantially whensingle-stranded to primers on IS1, wherein amplification generates alocalized clonal population of immobilized clonal amplicons around thepoint of initial hybridization of template 1 to IS1.

Also provided is a method of generating separated and immobilized clonalpopulations of a first template sequence (“template 1”) and a secondtemplate sequence (“template 2”), comprising amplifying the first andsecond template sequence to generate a population of clonal amplicons oftemplate 1 substantially attached to first immobilization site (“IS1”)and not to a second immobilization site (“IS2”), or a population ofclonal amplicons of template 2 substantially attached to IS2 and not toIS1, wherein: (a) both templates and all amplicons are contained withinthe same continuous liquid phase, where the continuous liquid phase isin contact with a first and second immobilization site (respectively,“IS1” and “IS2”), and where IS1 and IS2 are spatially separated, (b)template 1 when in single-stranded form comprises a first subsequence(“T1-FOR”) at one end, and a second subsequence (“T1-REV”) at itsopposite end, (c) template 2 when in single-stranded form comprises afirst subsequence (“T2-FOR”) at one end, and a second subsequence(“T2-REV”) at its opposite end, (d) IS1 comprises multiple copies of animmobilized nucleic acid primer (“IS1 primer”) that can hybridizesubstantially to T1-FOR and T2-FOR when T1 and T2 are single-stranded,(e) IS2 comprises multiple copies of an immobilized primer (“IS2primer”) that can hybridize substantially to both T1-FOR and T2-FOR whenT1 and T2 are single-stranded, (f) the reverse complement of T1-REV whensingle-stranded cannot hybridize substantially to primers on IS1, butcan hybridize substantially to a non-immobilized primer (“SP1”) in thecontinuous liquid phase; and (g) the reverse complement of T2-REV whensingle-stranded cannot hybridize substantially to primers on IS2, butcan hybridize substantially to a non-immobilized primer (“SP2”) in thecontinuous liquid phase.

In addition, a method is provided of generating separated andimmobilized clonal populations of a first template sequence (“template1”) and a second template sequence (“template 2”), comprising amplifyingthe first and second template sequence, wherein: (a) both templates arein single-stranded form and are both contained within the samecontinuous liquid phase, where a first and second immobilization site(respectively, “IS1” and “IS2”) are in contact with said continuousliquid phase, and where IS1 and IS2 are spatially separated, (b)template 1 comprises a first subsequence (“T1-FOR”) at its 3′ end, and asecond subsequence (“T1-REV”) that is non-overlapping with T1-FOR and atits 5′ end, (c) template 2 comprises a first subsequence (“T2-FOR”) atits 3′ end, and a second subsequence (“T2-REV”) that is non-overlappingwith T2-FOR and at its 5′ end, (d) IS1 comprises an immobilized primer(“IS1 primer”) that can hybridize to both T1-FOR and T2-FOR, (e) IS2comprises an immobilized primer (“IS2 primer”) that can hybridize toboth T1-FOR and T2-FOR, and (f) the reverse complement of T1-REV cannothybridize substantially to primers on IS1, and/or the reverse complementof T2-REV cannot hybridize substantially to primers on IS2, but can eachhybridize substantially to a non-immobilized primer in the continuousliquid phase; whereby amplification results in a population of clonalamplicons of template 1 substantially attached to IS1 and not to IS2,and/or a population of clonal amplicons of template 2 substantiallyattached to IS2 and not to IS1.

Optionally, in any method described herein, the continuous medium isflowable. Optionally, intermixing of non-immobilized nucleic acidmolecules is substantially unretarded in the continuous liquid phaseduring at least a portion of the amplification process, e.g., during anyone or more steps or cycles described herein.

Optionally, in any method described herein, intermixing is substantiallyunretarded for a period of time during amplification. For example,intermixing is substantially unretarded during the entire duration ofamplification.

Optionally, in any method described herein, any nucleic acid that hasdissociated from one immobilization site is capable of substantiallyhybridizing to both immobilization sites and any movement (e.g.,movement by diffusion, convection) of said dissociated nucleic acid toanother immobilization site is not substantially retarded in thecontinuous liquid phase.

Optionally, in any method described herein, the continuous liquid phaseis in simultaneous contact with IS1 and IS2.

Optionally, in any method described herein, a first portion of atemplate that is bound by an immobilized primer does not overlap with asecond portion of the template whose complement is bound by anon-immobilized primer.

Optionally, in any method described herein, at least one template to beamplified is generated from an input nucleic acid after the nucleic acidis placed in contact with at least one immobilization site.

Optionally, any method described herein comprising the steps of: (a)contacting a support comprising immobilized primers with asingle-stranded nucleic acid template, wherein: hybridizing a firstimmobilized primer to a primer-binding sequence (PBS) on the template(b) extending the hybridized first primer in template-dependentextension to form an extended strand that is complementary to thetemplate and at least partially hybridized to the template; (c)partially denaturing the template from the extended complementary strandsuch that at least a portion of the PBS is in single-stranded form(“free portion”); (d) hybridizing the free portion to a non-extended,immobilized second primer (e) extending the second primer intemplate-dependent extension to form an extended strand that iscomplementary to the template (f) optionally, separating the annealedextended immobilized nucleic acid strands from one another.

Optionally, in any method described herein (a) during amplification,nucleic acid duplexes are formed comprising a starting template and/oramplified strands; which duplexes are not subjected during amplificationto conditions that would cause complete denaturation of a substantialnumber of duplexes.

Optionally, in any method described herein, the single-strandedtemplates are produced by taking a plurality of input double-stranded orsingle-stranded nucleic acid sequences to be amplified (which sequencemay be known or unknown) and appending or creating a first universaladaptor sequence and a second universal adaptor sequence onto the endsof at least one input nucleic acid; wherein said first universal adaptorsequence hybridizes to IS1 primer and/or IS2 primer, and the reversecomplement of said second universal adaptor sequence hybridizes to atleast one non-immobilized primer. The adaptors can be double-stranded orsingle-stranded.

Optionally, in any method described herein, first and second nucleicacid adaptor sequences are provided at first and second ends of saidsingle-stranded template sequence.

Optionally, in any method described herein, a tag is also added to oneor more nucleic acid sequences (e.g., a template or a primer or anamplicons), said tag enabling identification of a nucleic acidcontaining the tag.

Optionally, in any method described herein, all primers on at least oneimmobilization site or support have the same sequence. Optionally, animmobilization site or support comprises a plurality of primers havingat least two different sequences. Optionally, two or more differenttypes of primer (e.g., different in sequence) are present insubstantially the same concentrations as one another, or alternativelyin different concentrations. Optionally, the primers of at least oneimmobilization site or support are substantially homogeneously dispersedover the immobilization site or support. Optionally, in any methoddescribed herein, two different immobilization sites are spatiallyseparated subcomponents of a single support and/or are on differentunconnected supports.

Optionally, in any method described herein, the support forms athree-dimensional matrix and the two different immobilization sites aretwo different three-dimensional portions of the support that are notcompletely overlapping. Optionally, in any method described herein, thetwo different immobilization sites are two different areas on thesurface of a support that are not completely overlapping. Optionally, inany method described herein, the two different immobilization sites areon different supports.

At least one support can be a bead, e.g., a microbead or nanobead. In anembodiment, the bead is a “scaffolded nucleic acid polymer particle” orSNAPP, described in U.S. Publ. App. No. 2010-0304982, incorporated byreference.

Optionally, in any method described herein, at least one immobilizationsite includes the entire surface of the support or the entire volume ofthe support. Optionally, in any method described herein, the twodifferent immobilization sites are located in a predeterminedarrangement (e.g. in a grid pattern). In other embodiments, one or moreimmobilization sites are not predetermined (e.g., the support comprisesimmobilized primers at non-predetermined locations), and the primer towhich a starting template hybridizes (e.g., before any amplificationoccurs) can be considered to be the immobilization site for thattemplate.

Optionally, in any method described herein, heating is used to partiallyseparate annealed nucleic acid strands. Optionally, primer extension isachieved by hybridizing the primer to a template, and contacting with apolymerase and nucleotides. The contacting and hybridizing can beachieved simultaneously or sequentially. Optionally, one or morenucleotides are detectably labeled.

Optionally, in any method described herein, the method further includesthe step of treating one or more extended immobilized nucleic acidstrands so as to release a nucleic acid molecule or a part thereof. Thetreating for example can optionally consist of nucleic acid cleavage,e.g., with a restriction endonuclease or with a ribozyme. For example,one or more of said primers has a restriction endonuclease recognitionsite or a ribozyme recognition site or has part of such a site, whichpart becomes complete when primer extension occurs.

Optionally, in any method described herein, the method is used toamplify a plurality of different nucleic acid sequences, e.g.,sequentially or simultaneously. The plurality is for example more than10³, 10⁵, 10⁷, 10⁹, 10¹¹, 10¹⁴, or 10²⁰ target nucleic acids.

Any method herein can be used to provide amplified nucleic acidmolecules for diagnosis or for screening or for genotyping, or toprovide amplified nucleic acid molecules to be used as a support forother components, or to generate additional nucleic acid molecules infree (e.g., non-immobilized rather than immobilized) form. For exampleany method can be used to monitor gene expression, or to identifynucleic acid molecules with gene products that are rarely expressed,identifying heterozygous individuals, nucleic acid fingerprinting.

Optionally, in any method described herein, said different nucleic acidsequences are each provided with a first and second nucleic acid“adaptor” sequence as described anywhere herein, said first and second“adaptor” sequences being the same for the each of the different nucleicacid sequences.

Optionally, in any method described herein, said different nucleic acidsequences are each provided with a different tag so that the differentsequences can be distinguished from one another.

In an embodiment, amplification is achieved using RPA, i.e.,recombinase-polymerase amplification (see, e.g., WO2003072805,incorporated by reference herein). RPA optionally is carried out withoutsubstantial variations in temperature or reagent conditions. In anembodiment herein, partial denaturation and/or amplification, includingany one or more steps or methods described herein, can be achieved usinga recombinase and/or single-stranded binding protein. Suitablerecombinases include RecA and its prokaryotic or eukaryotic homologues,or functional fragments or variants thereof, optionally in combinationwith single-strand binding proteins (SSBs). In an embodiment, therecombinase agent optionally coats single-stranded DNA (ssDNA) such asan amplification primer to form a nucleoprotein filament strand whichinvades a double-stranded region of homology on a template. Thisoptionally creates a short hybrid and a displaced strand bubble known asa D-loop. In an embodiment, the free 3′-end of the filament strand inthe D-loop is extended by DNA polymerases to synthesize a newcomplementary strand. The complementary strand displaces theoriginally-paired partner strand of the template as it elongates. In anembodiment, one or more of a pair of amplification primers are contactedwith one or more recombinase agents before be contacted with a templatewhich is optionally double-stranded.

In any method described herein, amplification of a template (targetsequence) comprises contacting a recombinase agent with one or more ofat least one pair of amplification primers, thereby forming one or more“forward” and/or “reverse” RPA primers. Any recombinase agent that hasnot associated with the one or more primers is optionally removed.Optionally, one or more forward RPA primers are then contacted with atemplate strand, which optionally has a region of complementarity to atleast one RPA primer. The template strand can be hybridized to aContacting of a RPA primer with a complementary template optionallyresults hybridization between said primer and the template. Optionally,the the 3′ end of the primer is extended along the template with one ormore polymerases (e.g., in the presence of dNTPs) to generate a doublestranded nucleic acid and a displaced template strand. The amplificationreaction can comprise repeated cycles of such contacting and extendinguntil a desired degree of amplification is achievable. Optionally thedisplaced strand of nucleic acid is amplified by a concurrent RPAreaction. Optionally, the displaced strand of nucleic acid is amplifiedby contacting it in turn with one or more complementary primers; and (b)extending the complementary primer by any strategy described herein.

In an embodiment the one or more primers comprise a “forward” primer anda “reverse” primer. Placing both primers and the template in contactoptionally results in a first double stranded structure at a firstportion of said first strand and a double stranded structure at a secondportion of said second strand. Optionally, the 3′ end of the forwardand/or reverse primer is extended with one or more polymerases togenerate a first and second double stranded nucleic acid and a first andsecond displaced strand of nucleic acid. Optionally the second displacedstrand is at least partially complementary to each other and canhybridize to form a daughter double stranded nucleic acid which canserve as double stranded template nucleic acid in a subsequentamplification cycles.

Optionally said first and said second displaced strands is at leastpartially complementary to said first or said second primer and canhybridize to said first or said second primer.

In an alternative embodiment of any method or step or composition orarray described herein, the support optionally comprises immobilizedprimers of more than one sequence. After a template nucleic acid strandhybridizes to first complementary immobilized primer, the first primercan then be extended and the template and the primer can be separatedpartially or completely from one another. The extended primer can thenbe annealed to a second immobilised primer that has different sequencefrom the first, and the second primer can be extended. Both extendedprimers can then be separated (e.g., fully or partially denatured fromone another) and can be used in turn as templates for extension ofadditional immobilized primers. The process can be repeated to provideamplified, immobilised nucleic acid molecules. In an embodiment, thisamplification results in immobilized primer extension products of twodifferent sequences that are complementary to each other, where allprimer extension products are immobilized at the 5′ end to the support.

The amplified nucleic acids generated from any method herein can be usedfor many different purposes, including sequencing, screening, diagnosis,in situ nucleic acid synthesis, monitoring gene expression, nucleic acidfingerprinting, etc.

Optionally, the template concentration is adjusted such that immobilizedtemplates are generally spaced as a sufficient distance from each otherthat the clonal clusters generated from individual templates have littleor substantially no overlap with each other, or do not contaminate oneanother during or after amplification or replication. Optionally, any ofthe amplification methods herein involve a step of adjusting theconcentration of template nucleic acid before it is contacted with asolid support so that individual template molecules hybridize to primersimmobilized on the solid support at a density of at least 10000, 100000,400000, 500000, 1,000,000 or 10⁷ molecules per mm². Optionally,individual template molecules are amplified in-situ on the support,giving rise to clonal populations that are spatially-centered around thepoint of hybridization of the initial template.

Strand Flipping

In a “flipping” embodiment described below, two or more primers areextended to form two or more corresponding extended strands. Optionally,the two or more primers that are extended comprise or consistessentially of substantially identical sequence, and the extendedportions of corresponding extended strand are at least partlynon-identical and/or complementary to each other.

One exemplary embodiment of flipping is as follows. A starting templateis amplified, e.g., by template walking, to generate a plurality ofprimer-extended strands (which for convenience will be designated as“forward” strands). Optionally, the forward strands are complementary tothe starting template. Optionally, the forward strands are immobilizedon the support. Optionally, the forward strands comprise substantiallyidentical sequence, e.g., the forward strands are substantiallyidentical to each other. In an embodiment the forward strands are formedby extension of one or more primers immobilized on a support (“forward”primers). The forward primers and/or the forward strands are optionallyattached to the support at or near their 5′ ends. Optionally, one ormore of the primer-extended forward strands comprises a 3′ sequence(called a self-hybridizing sequence) that is absent in the unextendedprimer and can hybridize under the conditions of choice to a 5′ sequence(this process will be termed “self-hybridization”). The 5′ sequence isoptionally part of the unextended forward primer. In an example, theforward extension product forms a “stem-loop” structure upon suchhybridization. Optionally, the unextended forward primer comprises a“cleavable” nucleotide at or near its 3′ terminus that is susceptible tocleavage. In an embodiment, the cleavable nucleotide is linked to atleast one other nucleotide by a “scissile” internucleoside linkage thatcan be cleaved under conditions that will not substantially cleavephosphodiester bonds.

After extension, the forward-primer extension product (i.e., the forwardstrand) is optionally allowed to self-hybridize. In a furtherembodiment, after allowing for self-hybridization the forward strand iscleaved at a scissile linkage of a cleavable nucleotide (for example anucleotide which forms a scissile linkage with a neighboringnucleotide). The cleavage results in two fragments of theprimer-extension product (i.e., the extended forward strand). In anembodiment, a first fragment comprises at least a portion of theoriginal unextended forward primer. Optionally, the first fragment doesnot comprise any extended sequence. Optionally, the first fragment isimmobilized (e.g., because the unextended forward primer was alreadyimmobilized). In an embodiment, a second fragment comprises extendedsequence. Optionally the second fragment comprises any 3′ portion of theunextended primer beyond the cleavable nucleotide or does not compriseany portion of the unextended primer. Optionally, the second fragment ishybridized to the first portion through its self-hybridizing sequence.

In an example, the cleavable nucleotide is one that is removed by one ormore enzymes. The enzyme can for instance be a glycosylase. Theglycosylase optionally has N-glycosylase activity which releases thecleavable nucleotide from double stranded DNA. Optionally, the removalof the cleavable nucleotide generates an abasic, apurinic orapyrimidinic site. The abasic site can optionally be further modified,for example by another enzymatic activity. Optionally, the abasic siteis modified by a lyase to generate a base gap. The lyase for examplecleaves 3′ and/or 5′ to the abasic site. Cleavage optionally occurs atboth the 5′ and 3′ end by the lyase, resulting in removing the abasicsite and leaving a base gap. Exemplary cleavable nucleotides such as5-hydroxy-uracil, 7, 8-dihydro-8-oxoguanine (8-oxoguanine),8-oxoadenine, fapy-guanine, methy-fapy-guanine, fapy-adenine, aflatoxinB1-fapy-guanine, 5-hydroxy-cytosine can be recognized and removed byvarious glycosylases to form an apurinic site. One suitable enzyme isformamidopyrimidine [fapy]-DNA glycosylase, also known as 8-oxoguanineDNA glycosylase or FPG. FPG acts both as a N-glycosylase and anAP-lyase. The N-glycosylase activity optionally releases damaged purinesfrom double stranded DNA, generating an apurinic (AP site), where thephosphodiester backbone is optionally intact. The AP-lyase activitycleaves both 3′ and 5′ to the AP site thereby removing the AP site andleaving a one-base gap. In an example the cleavable nucleotide is8-oxoadenine, which is coverted to a one-base gap by FPG with bothglycosylase and lyase activities.

In another embodiment the cleavable nucleotide is uridine. Optionally,the uridine is cleaved by “USER” reagent, which includes Uracil DNAglycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII, whereUDG catalyses the excision of a uracil base, forming an abasic(apyrimidinic) site while leaving the phosphodiester backbone intact,and where the lyase activity of Endonuclease VIII breaks thephosphodiester backbone at the 3′ and 5′ sides of the abasic site sothat base-free deoxyribose is released; after which kinase is optionallyused to convert the phosphate group on the 3′ end of cleaved product toan —OH group).

At least one cleaved fragment is optionally contacted with a polymerase.Optionally the first immobilized fragment can be extended by thepolymerase. If so desired the second hybridized fragment can act astemplate for extension of the first fragment. In an embodiment a“flipped” double-stranded extension product is formed. This flippedproduct can optionally be subjected to template walking in any mannerdescribed herein. When both flipped and unflipped are subjected totemplate walking, a cluster of two different extension products isformed, where both extension products have an identical portion(corresponding to the unextended primers) and portion complementary toeach other, corresponding to the extended portions of the extensionproducts.

In an embodiment, a sequence of interest, such as a self-hybridizingsequence or a new primer-binding site, can be optionally be added at the3′ ends of extended forward strands by contacting the extended forwardstrands with a single-stranded “splice” adaptor sequence in the presenceof extension reagents (e.g., a polymerase and dNTPs). This splicesequence optionally comprises a 3′ portion that is substantiallycomplementary to a 3′ end portion of the extended forward strand, and a5′ portion that is substantially complementary to the sequence ofinterest to be added. After hybridizing the splice adaptor to the 3′ endof the extended forward strand, the forward strand is subjected totemplate-dependent polymerase extension using the splice adaptor astemplate. Such extension results in the addition of the sequence ofinterest to the 3′ end of the extended forward strand.

Thus, any method of primer extension and/or amplification describedherein can include any one or more of the following steps:

-   -   a) extension of immobilized forward primers by template walking        to generate a plurality of extended forward strands which are        optionally identical;    -   b) optionally hybridizing a splice adaptor to a 3′ end of the        extended forward strands and subjecting the forward strands to        template-dependent extension using the splice adaptor as a        template, thereby adding a further 3′ sequence to the        further-extended forward strands, wherein a portion of the added        3′ sequence is complementary to a portion of the        unextendedforward primer and hybridizes thereto to form a        stem-loop structure    -   c) cleaving the forward strands at a scissile linkage of a        cleavable nucleotidelocated at or near the junction of        unextended forward primer sequence and extended forward strand        sequence; and optionally removing the cleavable nucleotide,        thereby generating two cleaved fragments, the first fragment        comprising a portion of an unextended forward primer hybridized        to a 3′ primer-complementary sequence on the second fragment;    -   d) optionally subjecting the first fragment to polymerase        extension using the second fragment as template to generate a        flipped forward strand;    -   e) optionally hybridizing a second splice adaptor to a 3′ end of        the flipped forward strand, and subjecting the forward strands        to template-dependent extension using the splice adaptor as a        template, thereby adding a further 3′ sequence to the flipped        forward strands, wherein a portion of the added 3′ sequence is a        new primer-binding sequence that is absent in the flipped        strands;    -   f) selectively extending or amplifying the flipped strands which        comprise the new primer-binding sequenceby contacting with the        new primer and extending or amplifying by any method, e.g., as        described herein. The new primer will not bind to unflipped        strands or to flipped strands that were not further extended in        step (e).

FIG. 8 shows a schematic depiction of an exemplary strand-flipping andwalking strategy. (A) Template walking, (B) Strand flipping to generateflipped strands, (C) addition of new primer-binding sequence Pg′ onfinal flipped strands.

I. Methods of Clonal Amplification

Overview

A nucleic acid is associated with (e.g., hybridized to) an appropriateprimer, which is optionally immobilized. The hybridized nucleic acid mayfor convenience be designated as the “template” strand or “reversestrand.” This word “template” is not intended to imply any particularfunctional, structural or sequence relationship with an input nucleicacid that was originally introduced into solution or with a finalnucleic acid product that is generated by the amplification process. Inan embodiment, a forward primer can hybridize to a first portion of thereverse strand. Another “reverse” primer is optionally present, which issubstantially identical to a second non-overlapping portion of thereverse strand. The two portions are for example non-overlapping if theydo not contain any subportions that are identical or complementary toeach other.

In an embodiment the template strand is optionally single-stranded overat least a portion that is complementary to the forward primer, whichportion is designated the forward “primer-binding sequence” or forwardPBS. The forward primer is optionally extended using the reverse strandas template to form an extended forward strand, resulting in a duplexbetween the hybridized template (reverse strand) and the forward strand.At least part of the primer portion of the forward strand can beseparated from the hybridized reverse strand. The forward strandcomprises a reverse PBS portion that can hybridize to a “reverse” primerand this reverse primer can in turn be extended to form an extendedreverse strand. Both the forward and reverse strand can then beseparated from each other and the process can be repeated to provide aclonal population of amplified, immobilized nucleic acid molecules.

Optionally, any one or more steps of separating forward and reversestrands from each other involves partial separation, e.g., separationthat dissociates a portion of the forward strand from a portion of thereverse strand, but does not abolish all association between the twostrands. Optionally, a portion of a forward strand is dissociated from areverse strand, while another portion of the same forward strand remainsassociated (e.g., by hybridization) with a reverse strand.

During partial separation, at least a portion of the forward PBS on thereverse strand is dissociated from the first forward strand. However,separation is “partial” because the forward strand and reverse strandremain associated with eachother overall. For example, another portionof the reverse strand remains hybridized to the forward strand.Optionally, the denatured portion of the reverse strand re-hybridizeswith a second forward primer. Thus a portion of the reverse strand ishybridized to the first forward strand, while another portion of thesame reverse strand is hybridized to the second forward primer. Thesecond forward primer is then extended along the reverse strand (usingthe reverse strand as template) to generate a second forward strand.Repeated cycles of amplification, where an amplification cycleoptionally comprises hybridization, =extension and (partial) separation,generate a clonal population of nucleic acids.

Optionally, one or more forward primers are immobilized on a supportwhich lacks any immobilized reverse primers, or vice versa. In anembodiment, the first and second forward primers are immobilized closetogether or adjacent to each other. The resulting clonal population ofnucleic acids comprises forward strands that are immobilized close to oradjacent to each other. In an embodiment, at least 10⁶, 10⁸, 10¹⁰, 10¹²,or 10¹⁴ primers are immobilized on a cm² or a cm³ of an individualsupport or immobilization site. Optionally, all forward primers areidentical in sequence, or have an identical 3′ portion.

In alternative embodiments, both the forward and/or reverse primer areoptionally immobilized on a support. Alternatively both primers arenon-immobilized.

Generally, a nucleic acid is clonally amplified onto a support on whichmultiple copies of a primer are immobilized. An exemplary nucleic acidis one of a collection of nucleic acids. Individual nucleic acids of thecollection for example can have one or more adaptor sequences at their5′ and/or 3′ ends and variable sequences in between, such as gDNA orcDNA. In an embodiment, the 3′ adaptor has a low T_(m) region (whereT_(m) is the temperature at which half of the DNA molecules are innon-denatured or double-stranded state and half are in denatured, e.g.,random coil, state), and the 5′ adaptor optionally has a higher Tmregion, or vice versa. The low T_(m) region is for example an A-rich,T-rich or pyrimidine-rich region, such as an AT (or U)-rich sequence,such as polyT, polyA, polyU and any combinations of A, T and U bases.Exemplary methods are described herein.

The methods described herein can be used for many different purposes,including sequencing, screening, diagnosis, in situ nucleic acidsynthesis, monitoring gene expression, nucleic acid fingerprinting, etc.

Non-limiting exemplary methods of clonal nucleic acid amplification on asupport are as follows.

A) Amplification on a Support

In some embodiments, the disclosed methods, compositions, systems,apparatuses and kits include nucleic acids (e.g., primers, templatesetc.) that are attached to a support. The nucleic acids can be attachedusing any suitable method. In some embodiments, the attachment betweenthe nucleic acid molecule and the support is mediated by covalentbonding, by hydrogen bonding (for example attachment of a templatenucleic acid to a support mediated by hybridization of the template toanother nucleic acid, e.g., primer, which is covalently attached to thesupport), Van Der Waal's forces, affinity interactions, and the like.Any suitable method for attachment of the nucleic acid sequence to thesupport can be used, including the use of binding pairs (e.g.,avidin/biotin; antigen/antibody). In some embodiments, one member of thebinding pair is attached to the support, the other member of a bindingpair is attached to the nucleic acid, and the nucleic acid is attachedto the support via interaction of the two members of the binding pair.

The support can be comprised of any material and have any dimensions orshape. The support can be selected to have properties or reactivitiesthat interfere only minimally with the amplification process. In someembodiments, the support is comprised of solid material; alternatively,it can be comprised at least partially of semi-solid, fluid or liquidmaterial. In some embodiments, the support is spherical, spheroidal,tubular, pellet-shaped, rod-shaped, octahedral, hexagonal, square ortrapezoidal in shape. In some embodiments, the support is porous. Insome embodiments, the support can be comprised of a hydrophilic porousmatrix such as a hydrogel. See, e.g., U.S. Patent Publication No.2010-0304982, Hinz et al.; and US Patent Publication No. 2010-0136544,Agresti et al.; all of which foregoing applications are incorporated byreference herein.

Among other things, novel methods of generating a localized clonalpopulation of immobilized clonal amplicons in or on a support areprovided. The support can for example be solid or semisolid. Theamplified clonal population is optionally immobilized to the support'sexternal surface or can also be within the internal surfaces of asupport (e.g., where the support is semisolid, e.g., with a gel ormatrix structure). Exemplary supports can be solid or semi-solid.Optionally, the semi-solid support comprises polyacrylamide, cellulose,polyamide (nylon) and cross-linked agarose, dextran and -polyethyleneglycol.

Optionally, the disclosed methods and compositions include theattachment of one or more individual members of a collection (e.g., acollection) of nucleic acids to one or more supports of a population ofsupports. For example, different nucleic acids of the collection can beattached to different supports. The resulting population of supportsincludes a plurality of supports each comprising a single nucleic acid.In some embodiments, the nucleic acids of the collection are doublestranded, and the collection is denatured to form a population ofsingle-stranded nucleic acids. In some embodiments, the support includesprimers, and one or more of the single stranded nucleic acids can beattached to the support through hybridization to primers on the surface.

Optionally before amplification the collection of nucleic acids can beappropriately diluted and contacted with the population of supports insolution, such that at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of thesupports (or immobilization sites where the population of supportsconsists of one or a few supports) become attached to no more than onenucleic acid. In some embodiments, the ratio of the number of nucleicacids to the total number of supports can be set to facilitatemono-clone formation by, e.g., maximizing the number of resultingsupports (or number of immobilization sites on a single support) thatinclude only a single nucleic acid, or choosing a ratio that isstatistically predicted to give more clonal supports (e.g., beads) thanlower or higher ratios.

Optionally, a single support is used in any of the amplification methodsherein, where the single support has a plurality of primers that canhybridize to the templates. In such an embodiment, the concentration ofthe template collection is adjusted before it is contacted with a solidsupport so that individual template molecules in the collection getattached or associated (e.g., by hybridization to primers immobilized onthe solid support) at a density of at least 10², 10³, 10⁴, 10⁵, 4×10⁵,5×10⁵, 6×10⁵, 8×10⁵, 10⁶, 5×10⁶ or 10⁷ molecules per mm².

Optionally, individual template molecules are amplified in-situ on thesupport, giving rise to clonal populations that are spatially-centeredaround the point of hybridization of the initial template. Optionally,the amplification generates no more than about 10², 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹⁵, or 10²⁰ amplicons from a singleamplified template. Optionally, the colonies of clonal amplicons aresituated on the solid support at a density of at least 10², 10³, 10⁴,10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 8×10⁵, 10⁶, 5×10⁶ or 10⁷ molecules per mm².

In some embodiments, the nucleic acid collection can be contacted withone or more supports under conditions where multiple nucleic acids bindto the same support. Such contacting can be particularly useful inmethods that involve parallel clonal amplification of nucleic acids indifferent regions of the same support. The ratio of the number ofnucleic acids to the surface area of the support can be adjusted tofacilitate mono-clone formation by, e.g., ensuring that the nucleicacids are appropriately spaced in the support to favor formation ofmonoclonal populations of amplified nucleic acids without substantialcross-contamination between different clonal populations. For examplewhere a single support us used, the collection of nucleic acids to beamplified is adjusted to such a dilution that the resulting amplifiedclonal populations generated from individual nucleic acids are generallydiscrete or distinct, e.g., without overlap. For example, individualnucleic acids within 50%, 70%, 80% or 90% or more of the amplifiedclonal populations are not interspersed with substantially non-identicalnucleic acids. Optionally, different amplified populations are not incontact or completely overlapping with other amplified populations, orare distinguishable from each other using a detection method of choice.

In some embodiments, the nucleic acids are attached to the surface of asupport. In some embodiments, the nucleic acids can be attached withinthe support. For example, for supports comprised of hydrogel or otherporous matrices, the nucleic acids can be attached throughout the volumeof the support including on the surface and within the support.

In some embodiments, the support (or at least one support in apopulation of supports) can be attached to at least one primer,optionally to a population of primers. For example, the support (or atleast one support) can include a population of primers. The primers ofthe population can be substantially identical each other, or may includea substantially identical sequence. One, some or all of the primers caninclude a sequence that is complementary to a sequence within one ormore nucleic acid templates. In some embodiments, the population ofprimers can include at least two noncomplementary primers.

The primers can be attached to the support through their 5′ end, andhave free 3′ ends. The support can be the surface of a slide or thesurface of a bead. The primers have low melting temperature, such asoligo (dT)₂₀ (SEQ ID NO:3), and can hybridize to the low T_(m) region ofthe collection adaptor. The distances between the primers need to beshorter than the adapter length to allow templates waking, oralternatively, a long primer with 5′ end long linker will increase thechance of walking.

In some embodiments, the support is attached to and/or contacted with aprimer and a template (or reverse strand) under conditions where theprimer and template hybridize to each other to form a nucleic acidduplex. The duplex can include a double stranded portion that comprisescomplementary sequences of the template and primer, where at least onenucleotide residue of the complementary sequences are base paired witheach other. In some embodiments, the duplex can also include a singlestranded portion. The duplex can also include a single stranded portion.The single stranded portion can include any sequence within the template(or primer) that is not complementary to any other sequence in theprimer (or template).

A non-limiting exemplary method of clonal nucleic acid amplification ona support is as follows. A nucleic acid (which shall be designated forconvenience as the reverse strand) is clonally amplified onto a supporton which multiple copies of a complementary forward primer are attached.An exemplary nucleic acid is one of a plurality of DNA collectionmolecules, that for example the plurality of nucleic acid members haveone or more common (“adaptor”) sequences at or near their 5′ and/or 3′ends and variable sequences in between, suoh as gDNA or cDNA. In anembodiment, the 3′ common portion, e.g., adaptor, has a breathable(e.g., low T_(m)) region, and the 5′ common sequence (e.g., adaptor)optionally has a less breathable (e.g., higher T_(m)) region, or viceversa. In another embodiment, both the 5′ and 3′ common sequences arebreathable. The breathable (e.g., low T_(m)) region is for example aregion that is rich in A, T and/or U, such as an AT (or U)-richsequence, such as polyT, polyA, polyU and any combinations of A, T and Ubases, or bases complementary to such bases. Exemplary methods aredescribed herein.

One non-limiting exemplary method of clonal nucleic acid amplificationon a support is shown in FIG. 1. A non-limiting description of anexemplary method is as follows.

A double stranded DNA library molecule is denatured and the singlestranded DNA is attached to the support through hybridization to theprimers on the surface. The ratio of number of DNA molecules to supportarea or number of beads is set to facilitate mono-clone formation.

Primers are attached on a support through their 5′ and have free 3′. Thesupport can be the surface of a slide or the surface of a bead. Theprimers have low melting temperature, such as oligo (dT)₂₀ (SEQ ID NO:3)or oligo (dA)₃₀ (SEQ ID NO:1) and can hybridize to the low Tm region ofthe library adaptor. The distances between the primers can be shorterthan the adapter length to allow templates waking, or alternatively, along primer with 5′ end long linker will increase the chance of walking.

A nucleic acid is clonally amplified onto a support on which multiplecopies of a primer are attached. An exemplary nucleic acid is one of aplurality of DNA library molecules, which for example have one or morecommon (e.g., “adaptor”) sequences at their 5′ and/or 3′ ends andvariable sequences in between, such as gDNA or cDNA. In an embodiment,the 3′ adaptor has a low Tm region, and the 5′ adaptor optionally has ahigher Tm region, or vice versa. The low Tm region is for example apyrimidine-rich region, such as an AT (or U)-rich sequence, such aspolyT, polyA, polyU and any combinations of A, T and U bases or basescomplementary to such bases. Exemplary methods are described herein.

B) Primer Extension

One or more primers, whether in soluble form or attached to a support,is incubated with a DNA polymerization or extension reaction mix, whichoptionally comprises any one or more of reagents such as enzyme, dNTPsand buffers. The primer (e.g., a forward primer) is extended.Optionally, the extension is a template-dependent extension of a primeralong a template comprising the successive incorporation of nucleotidesthat are individually complementary to successive nucleotides on thetemplate, such that the extended or nonextended forward primer iscomplementary to the reverse strand (also termed antiparallel orcomplementary). Optionally, the extension is achieved by an enzyme withpolymerase activity or other extension activity, such as a polymerase.The enzyme can optionally have other activities including 3′-5′exonuclease activity (proofreading activity) and/or 5′-3′ exonucleaseactivity. Alternatively, in some embodiments the enzyme can lack one ormore of these activities. In an embodiment the polymerase hasstrand-displacing activity. Examples of useful strand-displacingpolymerases include Bacteriophage Φ29 DNA polymerase and Bst DNApolymerase. Optionally, the enzyme is active at elevated temperatures,e.g., at or above 45° C., above 50° C., 60° C., 65° C., 70° C., 75° C.,or 85° C.

An exemplary polymerase is Bst DNA Polymerase (Exonuclease Minus), is a67 kDa Bacillus stearothermophilus DNA Polymerase protein (largefragment), exemplified in accession number 2BDP_A, which has 5′-3′polymerase activity and strand displacement activity but lacks 3′-5′exonuclease activity. Other polymerases include Taq DNA polymerase Ifrom Thermus aquaticus (exemplified by accession number 1TAQ), Eco DNApolymerase I from Echerichia coli (accession number P00582), Aea DNApolymerase I from Aquifex aeolicus (accession number 067779), orfunctional fragments or variants thereof, e.g., with at least 80%, 85%,90%, 95% or 99% sequence identity at the nucleotide level.

Generally, the extension step produces a nucleic acid, which comprises adouble-stranded duplex portion in which two complementary strands arehybridized to each other. In one embodiment, walking involves subjectingthe nucleic acid to partially-denaturing conditions that denature aportion of the nucleic acid strand but are insufficient to fullydenature the nucleic acid across its entire length. In an embodiment,the nucleic acid is not subjected to fully-denaturing conditions duringa portion or the entire duration of the walking procedure. As intendedherein, a nucleic acid molecule can be considered partially-denaturedwhen a portion of at least one strand of the nucleic acid remainshybridized to a complementary strand, while another portion is in anunhybridized state (even if it is in the presence of a complementarysequence). The unhybridized portion is optionally at least 5, 7, 8, 10,12, 15, 17, 20, or 50 nucleotides long. The hybridized portion isoptionally at least 5, 7, 8, 10, 12, 15, 17, 20, or 50 nucleotides long.

Optionally, a nucleic acid can be considered to be partially denaturedwhen a substantial fraction of individual molecules of the nucleic acid(e.g., above 20%, 30%, 50%, or 70%) are in a partially denatured state.Optionally less than a substantial amount of individual molecule arefully denatured, e.g., not more than 5%, 10%, 20%, 30% or 50% of thenucleic acid molecules in the sample. Similarly a nucleic acid isoptionally considered fully denatured when it lacks anydouble-strandedness (or lacks any hybridization to a complementarystrand) in more than 80% or 90% of individual molecules of the nucleicacid. Under exemplary conditions at least 50% of the nucleic acid ispartly denatured, but less than 20% or 10% is fully denatured. In othersituations, at least 30% of the nucleic acid is partly denatured, butless than 10% or 5% is fully denatured. Similarly, a nucleic acid can beconsidered to be non-denatured when a minority of “breathable” portionsof interest in the nucleic acid, e.g., a PBS, are denatured. Inexemplary annealing conditions at least 10%, 30%, 50%, 60%, 70%, 80% or90% of PBSs of nucleic acid molecules in the sample are hybridized tocorresponding primers.

In an embodiment, partially denaturing conditions are achieved bymaintaining the duplexes as a suitable temperature range. For example,the nucleic acid is maintained at temperature sufficiently elevated toachieve some heat-denaturation (e.g., above 45° C., 50° C., 55° C., 60°C., 65° C., or 70° C.) but not high enough to achieve completeheat-denaturation (e.g., below 95° C. or 90° C. or 85° C. or 80° C. or75° C.). Complete heat-denaturation conditions are for exampleconditions that would result in complete separation of a significantfraction (e.g., more than 10%, 20%, 30%, 40% or 50%) of a largeplurality of strands from their extended and/or full-length complements.In an embodiment the nucleic acid is subjected to isothermal conditions,where temperature variation is constrained within a limited range duringat least some portion of the amplification (e.g., the temperaturevariation is within 20° C., optionally within 10° C., for example within5° C., or 2° C.). Optionally, the temperature is maintained at or around50° C., 55° C., 60° C., 65° C., or 70° C. for at least about 10, 15, 20,30, 45, 60 or 120 minutes. Optionally, any temperature variation is notmore than 20° C., optionally within 10° C., for example within 5° C., or2° C. during one or more amplification cycles (e.g., e.g., 1, 5, 10, 20,or all amplification cycles performed). Optionally, thermocycling can beperformed (where temperature variance is within isothermal ornon-isothermal ranges). In an example, the temperature variation isconstrained between the denaturation step and another step such asannealing and/or extension. In an example, the difference between thedenaturation temperature and the annealing or extension temperature isnot more than 20° C., optionally within 10° C., for example within 5°C., or 2° C., for one or more cycles of amplification. The temperatureis for example constrained for at least 5, 10, 15, 20, 30, 35 orsubstantially all cycles of amplification.

Partial denaturation can also be achieved by other means, e.g., chemicalmeans using chemical denaturants such as urea or formamide, withconcentrations suitably adjusted, or using high or low pH (e.g., pHbetween 4-6 or 8-9). In an embodiment, partial denaturation andamplification is achieved using recombinase-polymerase amplification(RPA). Exemplary RPA methods are described herein.

In an embodiment, the sequence of the negative and/or positive stranddesigned such that a primer-binding sequence or a portion thereof isbreathable, i.e., is susceptible to denaturation under the conditions ofchoice (e.g., amplification conditions). The breathable portion isoptionally more susceptible than a majority of nucleic acids of similarlength with randomized sequence, or more susceptible than at leastanother portion of the strand comprising the breathable sequence.Optionally, the breathable sequence shows a significant amount ofdenaturation (e.g., at least 10%, 20%, 30%, 50%, 70%, 80%, 90% or 95% ofmolecules are completely denatured across the breathable sequence) atthe amplification conditions of choice. For example the breathablesequence is designed to be fully-denatured in 50% of strand molecules at30, 35, 40, 42, 45, 50, 55, 60, 65 or 70° C. under the conditions ofchoice (e.g., amplification conditions).

Optionally, the T_(m) of a nucleic acid strand (e.g., a primer ortemplate strand) is the temperature at which at least a desired fractionof a clonal population of duplexes are rendered completelysingle-stranded under the chosen reagent conditions, where an individualduplex comprises the nucleic acid strand in question hybridized to itsfull-length complement. By default, the desired fraction is 50% if thefraction is not specified. In alternative embodiments, the desiredfraction is optionally at least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 99%. Also for example a sequence can beconsidered breathable if the theoretically-predicted melting temperatureof the breathable sequence is not more than 20, 30, 35, 40, 45, 50, 55,60 or 65° C. under the amplification conditions of choice, using knowntheoretical calculations for predicted melting temperature (T_(m)). Inan example, the thermal stability, melting behavior and/or T_(m) is atheoretically-predicted temperature according to the teachings ofBreslauer et al., Proc. Nat. Acad. Sci. 83, 3746-50 (1986). In anexemplary calculation, T_(m) is predicted as follows:

$T_{m} = {\frac{\Delta\; H\frac{kcal}{C*{Mol}}}{{\Delta\; S} + {R\mspace{14mu}{\ln\left( {\lbrack{primer}\rbrack/2} \right)}}} - {273.15{^\circ}\mspace{14mu}{C.}}}$

where ΔH is the enthalpy of base stacking interactions adjusted forhelix initiation factors; ΔS is the entropy of base stacking adjustedfor helix initiation factors, and for the contributions of salts to theentropy of the system, and R is the universal gas constant (1.987 Cal/°C.*Mol). Further details and assumptions are set forth in SantaLucia, J.(1998) Proc. Nat. Acad. Sci. USA 95, 1460); Rychlik, W. and Rhoads, R.E. (1989) Nucl. Acids Res. 17, 8543; and Borer P. N. et al. (1974) J.Mol. Biol. 86, 843.

In another embodiment the T_(m) is empirically measured by knownmethods. (e.g., Spink, Methods Cell Biol. 2008; 84:115-41; Meunier-Prestet al., Nucleic Acids Res. 2003 Dec. 1; 31(23): e150; Brewood et al.,Nucleic Acids Res. 2008 September; 36(15): e98.)

In an embodiment, at least one PBS on each strand is breathable—e.g.,the forward PBS and the reverse PBS are both breathable. Optionally, anucleic acid such as a forward or reverse strand comprises twobreathable sequences. For example, a 5′ portion and a 3′ portion can bebreathable.

Where partial denaturation is achieved by heating or elevatedtemperatures, an exemplary breathable PBS may be pyrimidine-rich (e.g.,with a high content of As and/or Ts and/or Us). The PBS comprises forexample a poly-A, poly-T or poly-U sequence, or a polypyrimidine tract.One or more amplification or other primers (e.g., an immobilized primer)are optionally designed to be correspondingly complementary to theseprimer-binding sequences. An exemplary PBS of a nucleic acid strandcomprises a poly-T sequence, e.g., a stretch of at least 10, 15, 20, 25or 30 thymidine nucleotides, while the corresponding primer has acomplementary sequence to the PBS, e.g., a stretch of at least 10, 15,20, 25 or 30 adenosine nucleotides. Exemplary low-melt primersoptionally have a high proportion (e.g., at least 50%, 60%, 65%, 70%,75%, 80%, 85% 90% 95% or 100%) of nucleobases that generally (e.g.,under amplification conditions of choice) form no more than two hydrogenbonds with a complementary base when the primer is hybridized to acomplementary template. Examples of such nucleobases include A(adenine), T (thymine) and U (uracil). Exemplary low-melt primersoptionally have a high proportion of any one or more of A (adenine), T(thymine) and/or U (uracil) nucleotides or derivatives thereof. In anembodiment, the derivatives comprise nucleobases that are complementaryto A (adenine), T (thymine) and/or U (uracil). The portion of the primerthat hybridizes to the PBS optionally has at least 50%, 60%, 65%, 70%,75%, 80%, 85%, 90% 95% or 100% of A (adenine), T (thymine) or U (uracil)nucleotides, or any combination thereof. In another example, the portionof the primer that hybridizes to the PBS comprises a polyA sequence(e.g., at least 5, 10, 15, 20, 25 or 30 nucleotides long). Otherexemplary primers comprise (NA_(x))_(n) (SEQ ID NO:4) repeats.Optionally, n (in lower case) is from 2 to 30, e.g., from 3 to 10, forexample 4 to 8. “N” (in upper case) is any nucleotide—and optionally, Nis C or G. “A” is the shorthand convention for adenine, and “x” denotesthe number of adenine residues in the repeat, for example 2, 3, 4, 5, 6,10 or more. Exemplary primers comprise multiple repeats of (CAA)_(n)(SEQ ID NO:5), (CA)_(n) (SEQ ID NO:6), (CAAA)_(n) (SEQ ID NO:7) or even(GAA)_(n) (SEQ ID NO:8).

Optionally only one strand (e.g., the forward or the reverse strand) hasa breathable PBS. In another embodiment, both the forward and reversestrands have a breathable PBS. The breathable PBS is optionallycomplementary to a primer that is either immobilized to a support or isnot immobilized (e.g., in soluble form). Optionally the strandcomprising the breathable PBS is either immobilized to a support or isnot immobilized (e.g., in soluble form). Optionally both primers areimmobilized, or both strands are immobilized. Optionally neither primeris immobilized, or neither strand is immobilized.

An amplification cycle optionally comprises breathing, annealing andextension. The nucleic acid to be amplified is optionally subjected toconditions which are suitable for or optimized for at least one of thesesteps. In an embodiment, the nucleic acid is subjected to conditionswhich are suitable for more than one of these steps, (e.g., annealingand extension, or breathing and extension). In some instances, all threeof these steps can take place simultaneously under the same conditions.

In an exemplary method the nucleic acid can be subjected to conditionswhich permit or facilitate breathing. In an embodiment, “breathing” issaid to occur when the two strands of a double-stranded duplex aresubstantially hybridized to each other, but are denatured across a localportion of interest (e.g., the terminal ends or primer-binding sites).One or more breathable sequences (e.g., a forward and/or reverse PBShaving a low Tm portion) of the nucleic acid gets locally denatured(“breathes”) from a first complementary strand (e.g., a forward orreverse strand) which it is hybridized to, and is thus made available tohybridize to another second strand. An exemplary first strand is aprimer extension product from a first primer. An exemplary second strandis for example a second unextended primer (e.g., a PBS-complementaryoligonucleotide comprising, e.g., a dT or dA sequence). Optionally, thefirst and second strands are immobilized on a support, and can beclosely situated (e.g., in close enough proximity to allow walking). Theconditions for breathing are optionally partially-denaturing conditionsunder which the PBS is generally denatured but another portion ofnucleic acid remains in a hybridized or double-stranded state.Optionally, DNA helicase can be included in the reaction mix tofacilitate the partial denaturing.

Optionally, the nucleic acid is then subjected to conditions whichfacilitate annealing, e.g., the temperature is decreased, to enablehybridization between the breathable PBS and the second strand. In anembodiment, the same conditions are used to facilitate both breathingand extension. In another embodiment, annealing conditions are differentfrom breathing conditions—for example, the annealing conditions arenondenaturing conditions or conditions that favor denaturation less thanthe breathing conditions. In an example, annealing conditions involve alower temperature (such as 37° C.) than breathing conditions, in which ahigher temperature (e.g., 60-65° C.) is used. Optionally, fullydenaturing conditions are avoided during one or more cycles ofamplification (e.g., the majority of amplification cycles orsubstantially all amplification cycles).

Extension conditions are generally permissive or highly suitable forprimer extension. In an embodiment, the extension conditions of choiceare the same as or different from annealing and/or breathing conditions.In an embodiment, the same set of conditions is used for all three steps(e.g., isothermal amplification), such that subjecting the sample to asingle set of conditions for a sustained period enables multipleamplification cycles of breathing, annealing and extension to takeplace.

In an embodiment, strand extension is performed for example by a stranddisplacing DNA polymerase, such as Bst DNA polymerase large fragment,Klenow DNA polymerase, phi29 DNA polymerase, Vent DNA polymerase, anyfunctional fragments and/or variants, or any combination of suchenzymes. The strand-displacement capability optionally facilitatesextension through duplex portions of partially-denatured nucleic acids.

Optionally, one or more of the PBS-breathing and primer extension stepsare repeated multiple times to amplify an initial nucleic acid. Whereone or more nucleic acid reagents (e.g., primers) are immobilized to asupport, the primer-extension products remain substantially attached tothe support, e.g., by virtue of attachment of an unextended extendedprimer to the support prior to amplification, or by hybridization tosuch a primer). Optionally, a localized clonal population of clonalamplicons is formed around a discrete site on the support. An exemplarydiscrete site is a point of attachment of an initial nucleic acid strandto the support, and from which other nucleic acids within the clonalpopulation are directly or indirectly generated by primer extension,using the initial nucleic acid or its copies as a template.

Optionally, a sample is prepared of a population of one or more nucleicacids to be amplified. The population of nucleic acids can be insingle-stranded or double-stranded form; optionally one or more nucleicacids individually comprises a nucleic acid strand with a known 3′ endsequence and a known 5′ end sequence which are substantially identicalor complementary to the one or more primers used in the amplification. A3′ portion of the nucleic acid strand can for example be complementaryto an immobilized primer, whereas a 5′ portion can be identical to asoluble primer. The 5′ and/or 3′ portions can be common (“universal”) orinvariant between individual nucleic acids within the population.Optionally, the nucleic acids within the population individuallycomprise variant (e.g., unknown) sequence between the common portions,such as genomic DNA, cDNAs, mRNAs, mate-pair fragments, exomes. etc. Thecollection can for example have enough members to ensure over 50%, 70%,or 90% coverage of the corresponding genetice source (e.g., the genomeor the exome).

II. Compositions, Arrays and Kits

Also provided herein is a composition comprising any one or any subsetor all of the following: at least one reverse nucleic acid strand, aplurality of forward primers immobilized on at least one support, aplurality of reverse primers in solution, and a polymerase. The forwardand/or reverse primers are optionally low-melt or rich in adenine,thymine or uracil as described herein. An exemplary compositioncomprises clonal populations of nucleic acid strands (“reversestrands”), where individual reverse strands of each clonal populationcomprise a low-melt (e.g., breathable) primer-binding sequence at the 3′end and/or a low-melt primer sequence on the 5′ end. The compositionoptionally includes a plurality of reverse primers that aresubstantially identical to the low-melt primer sequence on a 5′ portionor end of the reverse strand. The composition optionally includes aplurality of forward primers that are substantially complementary to thelow-melt primer-binding sequence on a 3′ portion or end of the reversestrand. In an embodiment the forward primer and/or the reverse primer isimmobilized by attachment to a support. For example the forward primeris immobilized and the reverse primer is not immobilized, or vice versa.An exemplary composition comprises any one or more of: (1) a reversenucleic acid strand, (2) a plurality of low-melt forward primersimmobilized on a support, (3) a plurality of low-melt reverse primers insolution, and (4) a polymerase.

Optionally, the composition further comprises one or more extendedforward strands that are londer than unextended forward primers and areoptionally full-length complements of one or more reverse strands. In anembodiment one or more extended forward strands are hybridized to acomplementary reverse strand, where the reverse strand is optionallyalso hybridized to another different forward primer or to a differentforward strand. The different forward strand is optionally aless-than-full-length complement of the reverse strand. The compositioncan contain any one or more reagents described herein, and/or besubjected to any one or more procedures or conditions (e.g.,temperatures) described herein.

Optionally, the composition comprises a plurality of spatially-separatedclonal populations are attached to one or more solid supports. Forexample, a plurality of spatially-separated clonal populations areattached to the same support. The composition is optionally free ofanother enzyme that is not a polymerase, e.g., a recombinase or reversetranscriptase or helicase or nicking enzyme.

Optionally the composition comprises a collection of nucleic acidsproducible by any one or more methods described herein. For example, thecollection can comprise immobilized nucleic acids which occupy one ormore distinct areas on a surface, each area comprising a plurality ofidentical nucleic acid strands and optionally, a plurality of identicalcomplementary strands hybridized thereto, where the complementarystrands have no attachment or linkage or association with the solidsupport except by virtue of hybridization to the immobilized nucleicacid. Optionally, an individual nucleic acid strand within such an areais located so that another nucleic acid strand is located on the surfacewithin a distance of the length of that strand. Optionally there is atleast one distinct area present per mm² of surface on which the nucleicacids are immobilized. For example the number of distinct areas/mm² ofsurface on which the nucleic acids are immobilized is greater than 10²,greater than 10³, greater than 10⁴, greater than 10⁵, greater than 10⁶,greater than 10⁷, or greater than 10⁸.

The collections of amplified clonal populations can form arrays, whichcan be one-dimensional (e.g., a queue of generally monoclonalmicrobeads) or two-dimensional (e.g., the amplified clonal populationsare situated on a planar support), or three-dimensional. The individualclonal populations of an array are optionally but not necessarilysituated or arranged such that they are addressed or addressable.Optionally, different clonal populations are spaced at an appropriatedistance from one another, which distance is generally sufficient topermit different clonal populations to be distinguished from each other.In an embodiment, localized clonal populations are scattered in anordered or disordered, e.g., random, pattern over a planar substrate.

The features of an exemplary array are individual distinguishable clonalpopulations of nucleic acids, where optionally the features aredistributed over one or more supports. In an exemplary microbeadembodiment, an array comprises a plurality of microbeads, where anindividual microbead generally comprises a monoclonal population ofnucleic acids, and different microbeads generally comprise differentclonal populations (e.g., which differ in sequence). Optionally, themicrobeads are distributed or packed in a monolayer over a planarsubstrate. In other embodiments, the array comprises a single (e.g.,planar) support, the single support comprising a plurality of spatiallydiscrete clonal populations of nucleic acids, where different clonalpopulations optionally differ in sequence.

Optionally, one or more nucleic acids within individual clonalpopulations can be attached to the planar substrate directly. In anotherexample, the nucleic acids of individual clonal populations are attachedto microbeads, for example as discussed herein. The clonal microbeadsare optionally packed closely together over a planar substrate, inrandom or ordered fashion. Optionally, more than 20%, 30%, 50%, 70%,80%, 90%, 95% or 99% of the microbeads are in contact with at least one,two, four or six other microbeads. Optionally, less than 10%, 20%, 30%,50%, 70%, 80%, 90%, 95% or 99% of the microbeads are in contact withone, two, four or six other microbeads.

Optionally the features of an array (such as immobilization sites oramplified DNA clonal populations) are generally discrete or distinctfrom each other. For example, 50%, 70%, 80% or 90% or more of thefeatures of an array are not in contact or not completely overlappingwith other features on the same array, or are distinguishable from eachother using a detection method of choice. Optionally, the features canpartially overlap with each other as long as they remain distinguishablefrom each other.

Also provided herein are kits comprising any one or more reagents (e.g.,nucleic acids or enzymes or supports) described herein. For example akit can contain one or more primers, optionally immobilized. Anexemplary kit comprises two primers, where the denaturation temperatureof one primer is optionally at least 10, 20, 30 or 40° C. from theother. Optionally the lower-melting primer comprises aadenine/thymine/uracil-rich portion such as a polyA tract such as thosedescribed herein, e.g., a polyA, T or U sequence that is at least 10,15, 20, 25 or 30 nucleotides long. The lower-melting primer isoptionally immobilized, where the higher-melting primer is optionallynon-immobilized.

Optionally, the kit further contains one or more supports describedherein, comprising the immobilized primers. An exemplary supportcomprises a planar suface. Where multiple supports are used, the kit cancomprise microbeads bearing identical primers.

The kit optionally contains one or more polymerases, e.g., astrand-displacing polymerase, and/or any combination of amplificationreagents described herein.

The kit can also comprise instructions for diluting an initialpopulation of templates to be amplified, and/or a dilution medium.

III. Uses

The amplified and/or immobilized nucleic acid molecules generated frommethods described herein can be subjected to many differentapplications, including sequencing, screening, diagnosis, in situnucleic acid synthesis, monitoring gene expression, nucleic acidfingerprinting, forensics, diagnostics, etc.

Any method or plurality of nucleic acids described herein can be used inproviding nucleic acid molecules for sequence analysis. For example, oneor more nucleic acid molecules (e.g., amplicons) produced by any methoddescribed herein can be contacted with at least one sequencing analysisprimer and/or at least one probe. Optionally, at least one sequencinganalysis primer is or comprises an oligonucleotide. In an embodiment, atleast one probe comprises one or more nucleotides or one or moreoligonucleotides. Oligonucleotides used as sequence analysis primers orprobes for example can hybridize to at least one of the same sequencesas at least one immobilization primer, e.g., the oligonucleotidesoptionally have the same sequences as the immobilization primers.

In an embodiment, sequence analysis is performed by contacting one ormore target nucleic acid molecules to be analyzed with one or moresequence analysis primers and/or probes, removing any unhybridizedprobes and determining the label of the ligated probe.

In another embodiment, sequence analysis is performed by contacting oneor more target nucleic acid molecules to be analyzed with one or moresequence analysis primers and/or probes, and detecting any resultinghybridization between at least one target nucleic acid and at least onelabeled sequence analysis primer and/or probe. In an embodiment thesequence analysis method comprises extending one or more sequenceanalysis primers hybridized to target nucleic acid molecules by ligatingany adjacently-hybridized oligonucleotides probes to the sequenceanalysis primer, removing any unligated probes and determining the labelof the ligated probe.

In another embodiment, sequence analysis is performed by contacting oneor more target nucleic acid molecules to be analyzed with one or moresequence analysis primers and labeled nucleotide probes and atemplate-dependent polymerase, allowing the polymerase to incorporate alabeled nucleotide into a polymerase extension product of the sequenceanalysis primer, removing any unincorporated nucleotides, anddetermining the identity of the incorporated nucleotide. The nucleotideprobes are for example fluorescently-labeled, and/or the identity of theincorporated nucleotide is determined from its label. In anotherembodiment the nucleotide probe is not labeled and the presence orincorporation of the nucleotide into a polymerase extension product ismeasured by virtue of a by-product generated by incorporation. Forexample, incorporation of a nucleotide into an extension product isoptionally detected by measuring changes in pH or ions or an electriccurrent, for example by using a field-effect transistor.

In any method described herein, the probe (e.g., a nucleotide or anoligonucleotide) is optionally further extendable by polymerase orligase. Alternatively, the probe is optionally not extendable by apolymerase or ligase. Such non-extendable probes are optionally renderedextendable during the sequence analysis process (e.g., after theiridentity is determined). Optionally, nucleotide probes include A, G, C,T bases, where nucleotide probes with a different base at theinterrogation position of the probe have a different fluorescent label.

Optionally, all or less than all (e.g., at least one) sequencinganalysis primer or sequencing analysis probe comprises a labelednucleotides/oligonucleotides. Optionally, the labelednucleotides/oligonucleotides all have the same label or aredifferentially labeled. In an embodiment the labelednucleotides/oligonucleotides are fluorescence labeled. For example amixture of labeled and non-labeled nucleotides is used.

Optionally a plurality of different sequences are determined inparallel. For example, the sequence analysis is parallel sequenceanalysis of nucleic acid molecules present in at least 2, e.g., at least10, 1000, 1000, 106, 109 or 1012 different distinct areas. The sequenceanalysis technology can be polymerase-based sequence analysis, forexample Sanger sequence analysis. Examples of sequence analysistechnology include sequence analysis by synthesis, or sequence analysisusing reversible terminators, or ligation based sequence analysis, e.g.,two-base encoding using SOLiD.

Also provided is an apparatus for performing any method describedherein, comprising any one or more reagents or devices mentioned herein.For example the apparatus can comprise one or more supports, eachsupport comprising a plurality of immobilized primers (which areoptionally identical). For example the apparatus can comprise a nucleicacid polymerase or ligase, and/or a plurality of nucleotides (optionallylabeled) and means for partially separating annealed nucleic acidstrands. Exemplary means for separating annealed nucleic acid strandscomprises a controlled heating means, e.g., a heating means that canmaintain the reagents at a constant temperature. The apparatusoptionally comprises a source of one or more reactants described hereinand detector means for detecting one or more signals produced after oneor more of said reactants have been applied to said nucleic acidmolecules. The means for detecting optionally has sufficient resolutionto distinguish between the distinct immobilization sites of the support,or between multiple supports (if in the form of microbeads). Optionally,the apparatus comprises a support which has primers immobilized on aplanar surface.

In an embodiment the apparatus comprises a charge coupled device (CCD),which optionally is operatively connected with an imaging device.

In an embodiment each immobilization site is a separate bead, andwherein each bead comprises a clonal population of amplicons afteramplification. Optionally, each bead can be distributed into or on anarray before, during or after amplification. The array for example isany array of wells, where beads bearing amplicons are distributedindividually into separate wells. Optionally the array is a large-scaleFET array.

Optionally, at least some of the clonal populations generated by anymethod herein appear discrete from each other when detected or analyzed(e.g., by optical or electrochemical detection) and/or spatiallyseparated. In an embodiment where nucleotide incorporation is detectedby the generation of H+ ions, such ions can only diffuse a shortdistance, for example <100 nm in a buffer solution. This limitedcapacity for diffusion ensures H+ generated in one clonal populationwill generally not interfere with nearby clonal populations and willonly be significantly detectable by the sensor directly under the clonalpopulation.

The following examples are provided purely for illustrative purposes andare not intended to limit the scope of the present disclosure or claims.

EXAMPLES Example 1

5′-dual-biotin labeled oligo(dT)35 (SEQ ID NO:9) were bound to DynaBeadsMyOne streptavidin C1 magnetic beads. 80 million oligo(dT)35-bound beads(“(dT)35” disclosed as SEQ ID NO:9) and 800 ul DNA template at 0.01pg/ul were mixed in hybridization buffer. The DNA template sequence wasas follows:

(SEQ ID NO: 10) TTTTTTTTTTTTTTTTTTTT CCACTACGCCTCCGCTTTC CTC TCTATG GGC AGT CGG TGA TTC GTG GAA GAC GGG GGC AGTCTA TAC CCC TGT GGC GAC CAC TGC GCG GTG GTT TGCTAG GAG AGA ATG AGG AAC CCG GGG CAG.The following amplification protocol was performed to hybridize the DNAtemplates to the beads: 95° C. 2 min, 50° C. 1 min, 40° C. 1 min, 30° C.1 min, 25° C. 2 min. Beads were washed with washing buffer and theprimer was extended with Exo-Klenow by adding to the washed beads 10×NEBbuffer2 (40 ul), 25 mM dNTP, 4 ul, Exo-Klenow 40 u/ul, 4 ul, and water,352 ul, and incubating at room temperature for 10 minutes. The beadswere washed again and a template-walking reaction was performed asfollows. First, the following reaction mixture was prepared:

5 million beads

10× ThermoPol buffer 10 ul

25 mM dNTP 1 ul

P2 primer (CTG CCC CGG GTT CCT CAT TCT) (SEQ ID NO:11) 50 uM 2 ul

100×BSA 10 ul

Bst DNA polymerase large fragment 8 u/ul 10 ul

H2O 67 ul

This reaction mix was incubated at 60° C. for 45 minutes and 90 minuteswith shaking. Beads were washed and the amplified DNA on the beadsquantified with TaqMan qPCR, using the following reactants:

TaqMan forward primer:  (SEQ ID NO: 12) AGTCGGTGATTCGTGGAAGACTaqMan reverse primer:  (SEQ ID NO: 13) CTCATTCTCTCCTAGCAAACCACTaqMan probe:  (SEQ ID NO: 14) Fam-CCCCTGTGGCGACCAC-NFQ

The fold of amplification before and after the template walking reactionwas calculated and plotted against reaction time. Sufficientamplification for detection purposes was seen by 30 minutes or less.

Example 2

Binding of template to bead was done as described in Example 1, usingthe following template:

(SEQ ID NO: 10) TTTTTTTTTTTTTTTTTTTT CCACTACGCCTCCGCTTTC CTC TCTATG GGC AGT CGG TGA TTC GTG GAA GAC GGG GGC AGTCTA TAC CCC TGT GGC GAC CAC TGC GCG GTG GTT TGCTAG GAG AGA ATG AGG AAC CCG GGG CAG.

A template-walking reaction was performed as described in Example 1,except that 5 ul of 1 uM P2 primer (CTG CCC CGG GTT CCT CAT TCT) (SEQ IDNO:11) and 5 ul of 8 u/ul Bst DNA polymerase large fragment was added tothe reaction, and the beads were incubated at a range oftemperatures—specifically, 46° C., 51° C., 58° C., 60° C., 63° C., 65°C. for 45 minutes with shaking.

The DNA templates on the beads with TaqMan qPCR as described in Example1, using the following reagents:

TaqMan forward primer:  (SEQ ID NO: 12) AGTCGGTGATTCGTGGAAGACTaqMan reverse primer:  (SEQ ID NO: 13) CTCATTCTCTCCTAGCAAACCACTaqMan probe:  (SEQ ID NO: 14) Fam-CCCCTGTGGCGACCAC-NFQ

The delta Ct before and after the template walking reaction wascalculated and plotted against reaction temperature, as shown in FIG. 5.This figure shows the influence of temperature on the template walkingreaction.

Example 3

Oligo(dT)35 (SEQ ID NO:9) beads were prepared as in Example 1, and 80million oligo(dT)35-bound beads (“(dT)35” disclosed as SEQ ID NO:9) wereincubated with 800 ul of a mixture of two DNA templates (at equal molarratio) at 0.04 pg/ul in hybridization buffer. The DNA template sequenceis as following.

DNA template 1: (SEQ ID NO: 10)TTTTTTTTTTTTTTTTTTTT CCACTACGCCTCCGCTTTC CTC TCT ATG GGC AGT CGG TGA TTC GTG GAA GAC GGG GGC AGT CTA TAC CCC TGT GGC GAC CAC TGC GCG GTG GTT TGC TAG GAG AGA ATG AGG AAC CCG GGG CAG. DNA template 2: (SEQ ID NO: 15)TTTTTTTTTTTTTTTTTTTTCCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGATGAAACAGTTGATCATGGACAACCATATTCTGCTGTACGGCCAAGGCGGATGTACGGTACAGCAGATACTAAGATGATGAAGAGAATGAG GAACCCGGGGCAG.

Binding of template to beads and template-walking was performed asdescribed in Example 1, except that 20 ul of 8 u/ul Bst DNA polymeraselarge fragment was added, and beads were incubated at 60° C. for 45minutes with shaking. The beads were washed and diluted in a 96-wellqPCR plate to approximately 5 beads per well. 96 duplex TaqMan qPCRreactions were performed with the following primers and probes.

TaqMan forward primer1:  (SEQ ID NO: 12) AGTCGGTGATTCGTGGAAGACTaqMan reverse primer1:  (SEQ ID NO: 13) CTCATTCTCTCCTAGCAAACCACTaqMan probe1:  (SEQ ID NO: 14) Vic-CCCCTGTGGCGACCAC-NFQTaqMan forward primer2:  (SEQ ID NO: 16) GAAACAGTTGATCATGGACAACCATTaqMan reverse primer2:  (SEQ ID NO: 17) TCATCTTAGTATCTGCTGTACCGTACATTaqMan probe2:  (SEQ ID NO: 18) Fam-CCGCCTTGGCCGTACAG-NFQ

FIG. 6 shows the Ct values of the 96 duplex TaqMan qPCR reactions.

The following Table 1 summarizes the Ct values. The experimentalpercentages of beads populations are consistent with calculatedprobabilities based on monoclonal amplification model and Poisondistribution.

TABLE 1 Positive Probability 96 wells 100 copies cut off countspercentages (%) (%) FAM Ct <37 7.00 Vic Ct <34 8.00 FAM Ct <37 and VicCt <34 1.00 empty wells or beads 81.00 84.38 84.98 single template beads14.00 14.58 14.41 FAM and Vic 1.00 1.04 0.61

Example 4

Beads were prepared as in Example 1, except 5′-dual-biotin labeledoligo(dA)35 (SEQ ID NO:19) was used instead of Oligo(dt)35 (SEQ ID NO:9)as the bead-immobilized primer.

The following DNA template was bound to the prepared beads as describedin Example 1:

(SEQ ID NO: 20) AAAAAAAAAAAAAAAAAAAA CCACTACGCCTCCGCTTTC CTC TCTATG GGC AGT CGG TGA TTC GTG GAA GAC GGG GGC AGT CTA TAC CCC TGT GGC GAC CAC TGC GCG GTG GTT TGC TAG GAG AGA ATG AGG AAC CCG GGG CAG

The primer was not extended with Exo-Klenow. Template walking wasperformed as in Example 1 except that 20 ul of 120 u/ul Bst DNApolymerase large fragment was added and the beads incubated at 37 C for3 minutes and then at 60 C for 45 minutes and 90 minutes with shaking.The amplified DNA on the beads were quantified using TaqMan qPCR and thefollowing reagents:

TaqMan forward primer:  (SEQ ID NO: 12) AGTCGGTGATTCGTGGAAGACTaqMan reverse primer: (SEQ ID NO: 13) CTCATTCTCTCCTAGCAAACCACTaqMan probe: (SEQ ID NO: 14) Fam-CCCCTGTGGCGACCAC-NFQ

The fold of amplification before and after the template walking reactionwas calculated and plotted against reaction time, as shown in FIG. 7.After 90 minutes of template walking reaction, the DNA templates onbeads were amplified about 100,000 fold.

Example 5

5′-dual-biotin labeled oligo(dA)35 (SEQ ID NO:19) were bound toDynaBeads MyOne streptavidin C1 magnetic beads. 80 millionoligo(dA)35-bound beads (“(dA)35” disclosed as SEQ ID NO:19) were mixedwith 800 ul E coli DH10B genomic DNA fragment library at 0.1 pg/ul inhybridization buffer. Hybridization buffer was used as a negativecontrol. The DH10B fragment library had the following structure.

AAAAAAAAAAAAAAAAAAAA (SEQ ID NO:21)—P1 adaptor—150 by to 200 by DH10Bgenomic DNA fragments—P2 adaptor

The following program was run on a thermo cycler to hybridize the DNAtemplates to the beads: 95 C 2 min, 50 C 1 min, 40 C 1 min, 30 C 1 min,25 C 2 min. The beads were washed and a template walking reaction wasdone as follows:

5 million beads with DH10B library hybridized, or negative control beads

10× ThermoPol buffer 10 ul

25 mM dNTP 1 ul

P2 primer (CTG CCC CGG GTT CCT CAT TCT) (SEQ ID NO:11) 50 uM 2 ul

100×BSA 10 ul

Bst DNA polymerase large fragment 120 u/ul 10 ul

H2O 67 ul

The beads were incubated at 37 C for 3 minutes and then at 60 C for 45minutes with shaking. The beads were then washed and stained withfluorescent label by hybridizing a cy5 labeled oligonucleotide targetingthe P1 adaptor sequence in the DH10B fragment library. The stained beadswere laid on a microscope slide and imaged on a fluorescent microscope.

A shifted overlay of the white light image of the beads and the cy5fluorescent image of the beads revealed the white light images of thebeads as black dots and the cy5-labeled beads as red dots. About 15% ofthe beads were seen to be positively stained with cy5 and the rest werenegative.

Another exemplary protocol for template walking was performed asfollows:

Hybridization and primer extension: template was diluted in 1×NEB Buffer2 to a final concentration of 100 pM. Template was heated at 95° C. for3 min and then keep on ice. Hybridization/reaction mixes were preparedas follows:

100 pM denatured template: 48.5 μl 100 mM dNTP:  0.5 μl 5 U/μl Klenow:  1 μl Total volume:   50 μl

10 μl of reaction mixture was added to chosen lanes of StarLight flowcell and the flow cell at 37° C. for 20 minutes. Each lane was washedtwice with 1 ml 1×TMAX buffer

Template walking: A walking reaction mixture was prepared was follows:

10x NEB ThermoPol Buffer:  5 μl 100 mM dNTP:  1 μl 50 μM Primer Pe: 0.5μl  120 U/μl Bst (from NEB): 20 μl H₂O: 23.5 μl   Total volume: 50 μl

10 μl of reaction mixture was added to each lane, and the flow cellincubated at 60° C. for 20 minutes. Each lane was washed twice with 1 ml1×TMAX buffer, and the nucleic acid subjected to analysis as desired.

Example 6

This example describes clonal amplification of nucleic acids ontosupports in the form of matrices, followed by sequencing of theresulting amplified population using the Ion Torrent PGM™ sequencer,which is an ion-based sequencing system that detects byproducts ofnucleotide incorporation using a chemically sensitive field effecttransistor (chemFET). Exemplary embodiments are shown in FIG. 3. In anembodiment, at least one primer for template walking is immobilized on asurface (e.g., the floor) of individual wells, where an individual wellis operatively connected to a corresponding transistor.

Template Preparation: Libraries were prepared as described in the LifeTechnologies SOLiD 4.0 User Manual. Briefly, 2 ug of genomic DNAisolated from the E. coli strain DH10B was sheared using a Covaris S2system. The sheared DNA was end-repaired and was purified with the SOLiDLibrary Column Purification Kit.

A double-stranded adapter (“ION Adaptor 1”) was prepared by hybridizingthe single-stranded complementary oligonucleotides ION Adaptor Oligo 1and ION Adaptor Oligo 1′ to each other according to the procedure inAppendix C of the User Manual for the SOLiD™ 4 System (AppliedBiosystems, Life Technologies). ION Adaptor Oligo 1 included a primingsequence that was 30 mer long (i.e., was 30 nucleotides in length) andION Adaptor Oligo 1′ included the complement of this 30mer primingsequence at the 5′ end, plus an additional two thymidine residues (“TT”)at the 3′ end. Hybridization of ION Adaptor Oligo 1 with ION AdaptorOligo 1′ forms the double-stranded adaptor “Ion Adaptor 1”, whichincludes a 30-mer double-stranded priming sequence and has one blunt endand one “sticky” end including a 3′ overhang of two additional Tresidues. A third primer, Ion Adapter A PCR primer, which contained onlythe first 20 nucleotides of ION Adaptor Oligo 1, was used in the PCRstep as described below.

Similarly, a second double-stranded adaptor (“ION Adaptor 2”) wasprepared by hybridizing the complementary single-strandedoligonucleotides Ion T-Tailed Adapter Oligo 2 and Ion A-Tailed AdapterOligo 2′ using the procedure in Appendix C of the SOLiD 4.0 User Manual.ION T-tailed Adaptor Oligo 2 was 52mer in length and included a primingsequence of 30 nucleotides to the 5′ end, followed by a stretch of 22thymidine (“T”) residues (SEQ ID NO:22) at the 3′ end. ION A-tailedAdaptor Oligo 2′ was 50mer in length and included a stretch of 20adenine (“A”) residues (SEQ ID NO:21) at the 5′ end, followed by a30-nucleotide stretch complementary to the priming sequence of IONT-tailed Adaptor Oligo 2. Hybridization of the ION T-tailed AdaptorOligo 2 with the ION A-tailed Adaptor Oligo 2′ forms the double-strandedadaptor “Ion Adaptor 2”, which includes a 30mer double stranded primingsequence flanking a 20mer poly-A/poly-T duplex (SEQ ID NOS 21 and 3,respectively), and has one blunt end and one “sticky” end including a 3′overhang of two additional T residues.

The double-stranded adaptors Ion Adaptor 1 and Ion Adaptor 2 wereligated to the end-repaired DNA. The ligation product was size-selectedand subjected to 8 cycles of amplification using the following primers:Ion Adapter A PCR primer (which contained only the first 20 nucleotidesof ION Adaptor Oligo 1) and ION T-tailed Adapter Oligo 2. The amplifiedreaction product was subjected to size selection, yielding a finaladapted double-stranded DNA library with a median size of 209 bp.

Isothermal amplification of nucleic acids: The adapted double-strandedlibrary obtained as described above was mixed with polymer matricesincluding 30mer polyA primers (SEQ ID NO:1) (these matrices are referredto herein as “SNAPPs” for Scaffolded Nucleic Acid Polymer Particles, andwere prepared essentially as described in U.S. Patent Publication No.20100304982, Hinz et al.).

Annealing templates to particles: The double-stranded library wasannealed to the SNAPP particles. Briefly, 10 million SNAPPs were washed2× in buffer (each wash carried out by pipetting up and down toresuspend the SNAPPs, then pelleting the SNAPPs by centrifugation)

The dsDNA adapter-ligated and size-selected library was diluted inbuffer, such that the library was added at an average ratio of 1double-stranded template molecule per SNAPP, in a total volume of 100ul.

The template library was hybridized to the SNAPPs by running thefollowing thermocycling program:

-   -   95° C. for 3 minutes    -   50° C. for 1 minute    -   40° C. for 1 minute    -   30° C. for 1 minute    -   ° C. for 2 minutes

The template-annealed SNAPPs were washed twice in buffer.

Isothermal templating reaction and processing: The annealed and washedSNAPPs were resuspended in the following amplification reaction mixture:

1×Phi29 DNA Polymerase Reaction Buffer, 0.1 mg/mL BSA, 1 uM ION AdaptorA PCR primer, 1 mM equimolar dNTP mix and 100U Phi29 enzyme (NEB#M0269L)

The amplification reaction mixture including the SNAPPs were thenincubated in a thermomixer with 1000 RPM shaking as follows:

-   -   30° C. for 6 hours    -   65° C. for 30 minutes

The SNAPPS were pelleted and washed 2× in Life TEX Buffer

The double-stranded amplified DNA on the SNAPPs was then denatured toremove complementary strands that were not covalently attached to theSNAPP, by performing 2 washes in ION Denaturing Solution (125 mM NaOH,0.1% Tween-20) with a 5 minute incubation at room temperature for eachwash.

The SNAPPS were then washed 2× in Life TEX Buffer (10 mM Tris, 1 mMEDTA, 0.01% Triton X-100). The SNAPPs were then subjected to anion-based sequencing reaction using the Ion Torrent PGM™ sequencer, asdescribed below:

Sequencing Using the Ion Torrent Pgm™ Sequencer

The SNAPPs including the amplified DNA were washed 2× in AnnealingBuffer, which includes Gibco brand PBS, pH 7.2 (1.54 mM PotassiumPhosphate monobasic, 155.17 mM sodium chloride, 2.71 mM sodium phosphatedibasic) with 0.2% Tween-20.

5 ul of ION sequencing primer (Part number 602-1021-03, as provided inthe Nucleotides, Enzyme and Controls Kit, Part number 4462916, of theIon PGM™ 314 Sequencing Kit, Part No. 4462909; Ion Torrent Systems,which is a subsidiary of Life Technologies Corp., Carlsbad, Calif.) wasadded to the washed SNAPPs and hybridized in a thermocycler as follows:

-   -   95° C. for 2 minutes    -   37° C. for 2 minutes

Annealed SNAPPs were washed 2× in Annealing Buffer, with approximately 7ul of buffer left on the SNAPP pellet after the final wash.

1 ul of Ion Sequencing Polymerase (Part number 602-1023-01, as providedin the Nucleotides, Enzyme and Controls Kit, Part number 4462916, of theIon PGM™ 314 Sequencing Kit, Part No. 4462909; Ion Torrent Systems, asubsidiary of Life Technologies, Carlsbad, Calif.) was added to theSNAPPs and the mixture was incubated for 5 minutes at room temperature.

SNAPPs were gently sonicated for 10 seconds in a water bath to disperseany clumps.

2 ul of 50% glycerol (diluted in Annealing Buffer) was added and thesample was loaded on an Ion Torrent PGM™ 314 sequencing chip from theIon Sequencing 314 Kit (part number 4462909, Ion Torrent Systems, LifeTechnologies. Briefly, the chip was primed by addition of 50 ulAnnealing Buffer (part number 603-1047-01, included in the PGM ReagentsKit, part number 4465455, Ion Torrent Systems). 10 ul of sample wasinjected into the port of the chip, and the chip was then centrifugedfor 10 minutes. The PGM™ sequencing run was performed using standardnucleotide flow order for 55 cycles.

Using this method, a total of 21 alignments with a minimum of Q17quality (allows up to 2% error) were generated, representing 1391 mappedbases of DH10b sequence. Included in these sequencing reads were 18alignments that meet the criteria for Q20 quality (allows only 1%error), representing 956 mapped bases. The longest alignment obtainedwas 109 bases with Q20 quality (1% error, in this case 1 base mismatch).The longest perfectly mapped read (i.e. error-free read) was 95 baseslong.

Example 7 “Double” Walking

In this example, both the forward and reverse primers have a breathablePBS prone to local denaturation at the isothermal amplificationconditions of choice. Each PBS thus has a corresponding tendency toreassociate with a new unextended primer. The forward primer isimmobilized on a support, and thus the forward PBS, after dissociatingfrom a first forward primer, can rehybridize with a second differentforward primer that is immobilized close to the first forward primer.Similarly, the reverse PBS tends to dissociate from a first solublereverse primer and re-hybridize to a second soluble reverse primer,which is then extended.

The following amplification reaction was done:

Hybridization: template at 100 pM total concentration in 1×SSPEt buffer,and pre-denatured by heat (95° C. for 3 minutes) and held on ice. 12 ulwas added to each lane. The mix was subjected to 75° C. for 2 min, 50°C. for 1 min, 40° C. for 1 min, 30° C. for 1 min, 25° C. for 2 minutes.The lanes were washed with 0.1×SSPEt (2×1 ml).

Primer Extension: The following master mix was prepared: 5 μl of 10×NEBBuffer 2; 0.5 μl of 100 mM dNTP; 1 μl of Klenow (NEB 5 u/ul, M0212L);43.5 μl of dH2O (Total of 50 μl). 12 uL of reaction mix was added to alllanes, and incubated at 37 C for 30 min. Lanes were washed with2×SSC+0.1% SDS, 1×TMAX (1 ml) and incubated with 1×NEB ThermoPol bufferat 60° C. for 40 min and washed again with 2×SSC+0.1% SDS, 1×TMAX (1ml).

Template walking: The following master mix was prepared: 10 μl of 10×NEBThermoPol Buffer; 2 μl of 100 mM dNTP; 50 μl of soluble primer PE; 40 μlof Bst (NEB 120 u/ul); 47 μl of dH2O (Total of 100 μl). 12 μL, mastermix was added to each lane and incubated at 60° C. for 30 min. 12 μl ofthe supernatant was taken out and diluted 1:100, with 5 μl used forTaqman assays. Lanes were washed with 2×SSC+0.1% SDS, 1×TMAX (1 ml). Ina HiDi wash (2×12 uL for 5 min) the lanes were washed with 1×TMAX (2×1ml).

For the TaqMan reactions, a reaction nmix was prepared with 10 μl ofTaqMan Fast Universal Master Mix, No UNG; 1 μl of 20× primer probe mix;4 μl of H2O; 5 μl of Stock solution (Total: 20 μl). The mix wassubjected to 95° C. (20 sec) and 40 thermocycles (95° C. 3 sec, 60° C.30 sec), followed by data collection.

Amplification using two breathable primers (an immobilized “forward”primer and a soluble distal “reverse” primer, termed Pe or Pd in Table 2below) was more efficient than using one breathable primer alone.

TABLE 2 Average Cq Supernatant Release Copy/μm² Pc Conc. (uM) 0.8 20.214.9 334 Pd conc. (uM) 0.8 20.0 14.3 517 Pe Conc. (uM) 0.2 10.0 11.43357 0.3 8.3 11.4 3413 0.4 7.9 11.3 3601 0.5 7.8 10.9 4526

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description.

Unless otherwise apparent from the context, any feature can be claimedin combination with any other, or be claimed as not present incombination with another feature. A feature can be any piece ofinformation that can characterize an invention or can limit the scope ofa claim, for example any variation, step, feature, property,composition, method, step, degree, level, component, material,substance, element, mode, variable, aspect, measure, amount, option,embodiment, clause, descriptive term, claim element or limitation.

Generally, features described herein are intended to be optional unlessexplicitly indicated to be necessary in the specification. Non-limitingexamples of language indicating that a feature is regarded as optionalin the specification include terms such as “variation,” “where,”“while,” “when,” “optionally,” “include,” “preferred,” “especial,”“recommended,” “advisable,” “particular,” “should,” “alternative,”“typical,” “representative,” “various,” “such as,” “the like,” “can,”“may,” “example,” “embodiment,” or “aspect,” “in some,” “example,”“exemplary”, “instance”, “if” or any combination and/or variation ofsuch terms.

Any indication that a feature is optional is intended provide adequatesupport (e.g., under 35 U.S.C. 112 or Art. 83 and 84 of EPC) for claimsthat include closed or exclusive or negative language with reference tothe optional feature. Exclusive language specifically excludes theparticular recited feature from including any additional subject matter.For example, if it is indicated that A can be drug X, such language isintended to provide support for a claim that explicitly specifies that Aconsists of X alone, or that A does not include any other drugs besidesX. “Negative” language explicitly excludes the optional feature itselffrom the scope of the claims. For example, if it is indicated thatelement A can include X, such language is intended to provide supportfor a claim that explicitly specifies that A does not include X.

Non-limiting examples of exclusive or negative terms include “only,”“solely,” “consisting of,” “consisting essentially of,” “alone,”“without”, “in the absence of (e.g., other items of the same type,structure and/or function)” “excluding,” “not including”, “not”,“doesn't”, “cannot,” or any combination and/or variation of suchlanguage.

Similarly, referents such as “a,” “an,” “said,” or “the,” are intendedto support both single and/or plural occurrences unless the contextindicates otherwise. For example “a dog” is intended to include supportfor one dog, no more than one dog, at least one dog, a plurality ofdogs, etc. Non-limiting examples of qualifying terms that indicatesingularity include “a single”, “one,” “alone”, “only one,” “not morethan one”, etc. Non-limiting examples of qualifying terms that indicate(potential or actual) plurality include “at least one,” “one or more,”“more than one,” “two or more,” “a multiplicity,” “a plurality,” “anycombination of,” “any permutation of,” “any one or more of,” etc. Claimsor descriptions that include “or” between one or more members of a groupare considered satisfied if one, more than one, or all of the groupmembers are present in, employed in, or otherwise relevant to a givenproduct or process unless indicated to the contrary or otherwise evidentfrom the context.

Furthermore, it is to be understood that the inventions encompass allvariations, combinations, and permutations of any one or more featuresdescribed herein. Any one or more features may be explicitly excludedfrom the claims even if the specific exclusion is not set forthexplicitly herein. It should also be understood that disclosure of areagent for use in a method is intended to be synonymous with (andprovide support for) that method involving the use of that reagent,according either to the specific methods disclosed herein, or othermethods known in the art unless one of ordinary skill in the art wouldunderstand otherwise. In addition, where the specification and/or claimsdisclose a method, any one or more of the reagents disclosed herein maybe used in the method, unless one of ordinary skill in the art wouldunderstand otherwise.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. Genbank records referenced by GID or accession number,particularly any polypeptide sequence, polynucleotide sequences orannotation thereof, are incorporated by reference herein. The citationof any publication is for its disclosure prior to the filing date andshould not be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

Where ranges are given herein, the endpoints are included. Furthermore,it is to be understood that unless otherwise indicated or otherwiseevident from the context and understanding of one of ordinary skill inthe art, values that are expressed as ranges can assume any specificvalue or subrange within the stated ranges in different embodiments ofthe invention, to the tenth of the unit of the lower limit of the range,unless the context clearly dictates otherwise.

What is claimed:
 1. A method for amplifying a plurality of nucleic acidtemplates, comprising: a) providing a plurality of forward primersimmobilized on a support, wherein the plurality of forward primersincludes a first forward primer and a second forward primer, wherein theplurality of forward primers share at least 90% sequence identity, andwherein the distances between the first forward primer and the secondforward primer on the support is not more than twice the length of thefirst forward primer; b) providing a nucleic acid reverse strand fromthe plurality of nucleic acid templates, wherein the nucleic acidreverse strand includes a forward primer-binding sequence that canhybridize to any one of the plurality of forward primers; c) hybridizingthe first forward primer to the forward primer-binding sequence on thenucleic acid reverse strand; d) generating a first extended forwardstrand by primer extension of the first forward primer using the nucleicacid reverse strand as a template, wherein at least 85% of the firstextended forward strand residues are complementary to the nucleic acidreverse strand residues; e) performing a walking step, wherein thewalking step comprises denaturing at least a portion of the forwardprimer-binding sequence of the nucleic acid reverse strand from thefirst forward primer and hybridizing the second forward primer to theforward primer-binding sequence on the nucleic acid reverse strand,wherein a portion of the nucleic acid reverse strand is hybridized tothe first extended forward strand; f) generating a second extendedforward strand by primer extension of the second forward primer usingthe nucleic acid reverse strand as a template, wherein at least 85% ofthe second extended forward strand residues are complementary to thenucleic acid reverse strand residues; and g) amplifying the plurality ofnucleic acid templates simultaneously in a single continuous liquidphase, by performing one or more amplification cycles comprising steps(e) - (f) under isothermal conditions to form a plurality of clonalnucleic acid populations, wherein the second forward primer of step (e)of an amplification cycle acts as the first forward primer of step (e)of a subsequent amplification cycle, and wherein at least 50% of thenucleic acid strands or their complements in each clonal nucleic acidpopulation are at least 75% identical.
 2. The method of claim 1, whereinthe support comprises only primers having sequences that aresubstantially identical to the plurality of forward primers.
 3. Themethod of claim 1, wherein step (e) comprises: denaturing all of theforward primer-binding sequence on the nucleic acid reverse strand fromthe first extended forward strand and hybridizing the forwardprimer-binding sequence to the second forward primer.
 4. The method ofclaim 1, wherein at least 60% of the bases of the plurality of forwardprimers are adenine, thymine or uracil, or are complementary to adenine,thymine or uracil.
 5. The method of claim 1, wherein primer extension ofthe second forward primer using the nucleic acid reverse strand as atemplate results in displacing the first extended forward strand that ishybridized to the nucleic acid reverse strand.
 6. The method of claim 1,further comprising: hybridizing the forward primer-binding sequence onthe nucleic acid reverse strand to a third forward primer of theplurality of forward primers, wherein a portion of the nucleic acidreverse strand is also hybridized to the first or second extendedforward strand.
 7. The method of claim 6, wherein the hybridizing theforward primer-binding sequence on the nucleic acid reverse strand to athird forward primer comprises: denaturing all of the forwardprimer-binding sequence on the nucleic acid reverse strand from thesecond extended forward strand and re-hybridizing the forwardprimer-binding sequence to the third forward primer.
 8. The method ofclaim 7, further comprising: generating a third extended forward strandby primer extension of the third forward primer using the nucleic acidreverse strand as a template, wherein at least 85% of the secondextended forward strand residues are complementary to the nucleic acidreverse strand residues.
 9. The method of claim 5, further comprising:a) hybridizing a first reverse primer to a reverse primer-bindingsequence on the first or second extended forward strand, by denaturingat least a portion of the reverse primer-binding sequence on the firstor second extended forward strand from the nucleic acid reverse strand,wherein the first reverse primer is a soluble primer; b) extending thefirst reverse primer using the first or second extended forward strandas a template; and c) generating an extended reverse strand wherein atleast 85% of the extended reverse strand residues are complementary tothe first or second extended forward strand residues.
 10. The method ofclaim 1, wherein the amplifying is carried out by adjusting theisothermal conditions to a temperature range between 45° C. and 80° C.11. The method of claim 1, wherein the forward primers in the pluralityof forward primers are immobilized adjacent to each other in acontinuous lawn on the support.
 12. The method of claim 1, wherein theplurality of nucleic acid templates is produced by taking a plurality ofinput nucleic acid sequences and appending a first universal adaptorsequence onto the ends of at least one input nucleic acid sequence, andwherein the first universal adaptor sequence hybridizes to theimmobilized forward primers.
 13. The method of 1, wherein individualclonal nucleic acid populations are immobilized within or on a differentsupport from other amplified populations.
 14. The method of claim 9,further comprising subjecting the extended forward strands and theextended reverse strands to a sequencing reaction.
 15. The method ofclaim 10, wherein at least 50% of the nucleic acid reverse strands arehybridized to forward strands at all times during the amplifying. 16.The method of claim 10, wherein the amplifying is carried out using apolymerase that is active at a temperature at or above 45° C.
 17. Themethod of claim 16, wherein the amplifying is carried out by maintainingthe temperature at a range of between 45° C. and 70° C.
 18. The methodof claim 16, wherein the amplifying is carried out by maintaining thetemperature at or around 50° C., 55° C., 60° C., 65° C., or 70° C. forbetween 10 and 120 minutes.
 19. The method of claim 1, wherein theamplifying is carried out by adjusting the isothermal conditions to atemperature that is higher than the T_(m) of all forward primers butlower than the T_(m) of the nucleic acid reverse strands.
 20. The methodof claim 12, wherein the forward primers are separated from each otheron the support by a distance that is shorter than the length of theuniversal adapter.